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I N T E R N A T I O N A L SYMPOSIUM Modulating Ageing Anti-Ageing Heart Centre University Hospital Halle Ernst-Grube-Str. 40 06120 Halle (Saale) From Friday, September 1 st , 2017, 6 pm till Sunday, September 3 rd , 2017, 1:30 pm Opening: Lion Building Universitätsplatz 11, Halle (Saale) Conference Site: Steintor-Campus Adam-Kuckhoff-Straße 35, 06120 Halle (Saale) Keynote Speaker Claudio Franceschi Bologna, Italy from Molecular Biology to Clinical Perspectives German National Academy of Sciences Leopoldina German Society of Gerontology and Geriatrics (DGGG) Interdisciplinary Centre on Ageing Halle (IZAH) RTG 2155: ProMoAge
Transcript
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Modulating Ageing Anti-Ageing

Heart CentreUniversity Hospital Halle

Ernst-Grube-Str. 4006120 Halle (Saale)

From Friday,

September 1st, 2017, 6 pm

till Sunday,

September 3rd, 2017, 1:30 pm

Opening: Lion BuildingUniversitätsplatz 11, Halle (Saale)

Conference Site: Steintor-CampusAdam-Kuckhoff-Straße 35, 06120 Halle (Saale)

Keynote SpeakerClaudio FranceschiBologna, Italy

from Molecular Biology to Clinical Perspectives

German National Academy of Sciences Leopoldina

German Society of Gerontology and Geriatrics (DGGG)

Interdisciplinary Centreon Ageing Halle (IZAH)

RTG 2155: ProMoAge

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Modulating Ageing / Anti-Ageing:

from Molecular Biology to Clinical Perspectives

September 01st

– 03rd

2017

Heart Centre University Hospital Halle (Saale)

in cooperation with

German National Academy of Sciences Leopoldina

German Society of Gerontology and Geriatrics

(DGGG)

Interdisciplinary Centre on Ageing Halle (IZAH)

RTG 2155: ProMoAge

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Index

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4 Running Program

5 Abstracts Speakers

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Aging and diseases: common mechanisms but different rates Claudio Franceschi

Defining the target group: risk prediction of ageing trajectories Andrea Maier

Do biomarkers of ageing reflect real age? Andreas Simm

Challenges understanding aging through biomarkers across populations: the example of calcium Tamàs Fülöp

Motor and cognitive intervention to improve functional mobility and reduce falls in older adults and patients with neurodegeneration Anat Mirelman

Age-dependent posttranslational protein modification – driving force in neurodegeneration? Hans-Ulrich Demuth

Protein oxidation and aggregation in aging and senescence Tilman Grune

Proteasome modulation as an anti-aging and antiaggregation strategy Niki Chondrogianni

Glycated proteins in nutrition: friend or foe Katarina Sebekova

Cardiac ageing enhances atrial fibrillation or vice versa? – Impact of altered miRNA expressional profile and metabolic signalling for treatment and persistence of atrial fibrillation? Bernd Niemann

Protective effects of caffeine in the cardiovascular system depend on mitochondrial p27 – in aging and disease Judith Haendeler

Oral anticoagulation and polypharmacy in older adults with atrial fibrillation Markus Gosch

Gender related aspects of the cardiovascular system and its impact on progression and aging Sandra Eifert

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Historical overview of research on lifespan extension and its ethical implications Georg M. Martin

Rejuvenation biotechnology: why age may soon no longer mean aging Aubrey de Grey

The shape of lives to come Ian Richard Ground

Ethical aspects of biogerontological research Zoé Rheinsberg

Epigenetic stress response and stem cell aging Lenhard Rudolph

Understanding muscle stem cell regenerative decline with aging Pura Munoz-Cánove

Bioengineered matrices to regenerate the heart – The Zurich LifeMatrix programm Maximilian Emmert

Mesenchymal stem cells-on-chips/organ-on-chips/chips in mesenchymal organs – monitoring healing kinetics: an ageing perspective Günter Lepperdinger

Effects of pharmacological inhibition of aging-associated pathways on Drosophila lifespan Alexey Moskalev

The Path towards translating senolytic drugs and other interventions targeting aging processes into clinical treatments James Kirkland

Cellular senescence and aging Judith Campisi

DNA damage: impact on aging, neurodegeneration and the effect of nutritional interventions Jan Hoeijmakers

58 Posters ( in alphabetical order)

114 List of speakers

118

119

Organizers

Involved Societies

120 Authors Index

125 List of Sponsors

126 Map of Halle (Saale)

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R u n n i n g P r o g r a m

Modulating Ageing / Antiageing: From Molecular Biology to Clinical Perspectives

- Meeting language English -

Friday September 01st 2017 18:00 Opening

Andreas Simm

Address M. Gekle, Dean of the Medical Faculty,

Martin-Luther-University Halle-Wittenberg T. Klöss, Medical Director of the University Hospital Halle (Saale) H. Treede, Heart Centre of the University Hospital Keynote lecture and Schober award

Laudation Claudio Franceschi by Judith Campisi, Novato, USA

Keynote lecture: Claudio Franceschi University of Bologna, Bologna, I

“Aging and diseases: common mechanisms but different rates”

20:00 Come Together (Löwengebäude – Lion Building)

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Saturday September 02nd 2017

08:00 – 10:00 Session 1

Setting up the problem, predictors and markers Chair: Claudio Franceschi, Andreas Simm Defining the target group: risk prediction of ageing Andrea Maier trajectories Do biomarkers of ageing reflect real age? Andreas Simm Challenges understanding aging through biomarkers Tamás Fülöp across populations: the example of calcium Motor-Cognitive Interventions to reduce the risk of falls Anat Mirelman in older adults and neurodegenerative conditions

10:00 – 10:30 Coffee Break

10:30 – 12:30 Session 2

Protein damage and Aggregation Chair: Günter Lepperdinger, Lenhard Rudolph

Age-dependent posttranslational protein modification – Hans-Ulrich Demuth

driving force in neurodegeneration?

Protein Oxidation and Aggregation in Aging and Tilman Grune Senescence Proteasome modulation as an anti-aging and anti- Niki Chondrogianni aggregation strategy Glycated proteins in nutrition: friend or foe Katarina Sebekova

12:30 – 13:30 Lunch Break

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Saturday September 02nd 2017

13:30 – 15:30 Session 3

Cardiovascular System

Chair: Tamàs Fülöp, Maximilian Emmert Cardiac ageing enhances atrial fibrillation or vice versa? – Bernd Niemann Impact of altered miRNA expressional profile and metabolic signalling for treatment and persistence of atrial fibrillation? Protective effects of caffeine in the cardiovascular system Judith Haendeler depend on mitochondrial p27 - in aging and disease Oral anticoagulation and polypharmacy in older adults Markus Gosch with atrial fibrillation Gender related aspects of the cardiovascular system and Sandra Eifert its impact on progression and aging

15:30 – 16:30 Poster Session / Coffee Break

16:30 – 18:30 Session 4

Panel discussion on ethical issues Chair: Jutta Schnitzer-Ungefug Historical overview of research on lifespan extension George M. Martin and its ethical implications Rejuvenation biotechnology: Aubrey de Grey why age may soon no longer mean aging The shape of lives to come Ian Richard Ground Ethical aspects of biogerontological research Zoé Rheinsberg

20:00 Conference-Dinner (Halloren- und Salinemuseum)

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Sunday September 03rd 2017

08:30 – 10:30 Session 5

Stem Cells and Regeneration Chair: Judith Campisi, Judith Haendeler Epigenetic stress response and stem cell aging Lenhard Rudolph Understanding muscle stem cell regenerative decline Pura Munoz-Cánove with aging Bioengineered matrices to regenerate the heart – Maximilian Emmert The Zurich LifeMatrix programm Mesenchymal stem cells-on-chips/organ-on-chips/chips Günter Lepperdinger in mesenchymal organs – Monitoring healing kinetics: an ageing perspective

10:30 – 11:30 Poster Session / Coffee Break

11:30 – 11:45 Posterprice

11:45 – 13:45 Session 6

Anti-Aging Therapies Chair: Andrea Maier, George M. Martin Effects of pharmacological inhibition of aging-associated Alexey Moskalev pathways on Drosophila lifespan The path towards translating senolytic drugs and other James Kirkland interventions targeting aging processes into clinical treatments Cellular senescence and aging Judith Campisi DNA damage: impact on aging, neurodegeneration and Jan Hoeijmakers the effect of nutritional interventions

13:45 – 14:15 Farewell

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Aging and diseases: common mechanisms but different rates

Claudio Franceschi

The new GEROSCIENCE is based on the strong epidemiological evidence that aging is the

major risk factor for age-related diseases, and assumes that they share a common set of basic

mechanisms. This assumption is supported by a large panoply of experimental data and its

major consequence is that the primary target of Medicine is to combat aging instead of any

single age-related disease one by one, as favored by the present fragmentation into hundreds

of specialties and sub-specialties. If the same molecular and cellular mechanisms underpin both

aging and age-related diseases, a major question emerges: which is the difference between

aging and diseases? This is an old question that was addressed starting from Terentius and

Cicero who argued pros and cons, respectively, whether “senectus ipsa est morbus”, and

recently this topic was addressed by several scholars. On the basis of a variety of recent

experimental data and of the conceptualizations I proposed in the last years (the “remodeling”

theory of aging; inflammaging and garbaging; liquid immune self and immunobiography) I will

illustrate the hypothesis that age-related diseases - including the not so well-defined geriatric

syndromes and conditions such as frailty, metabolic syndrome and mild cognitive impairment -

can be conceptualized as ACCELERATED aging. Within such a perspective centenarians

represent one extreme (healthy aging/extreme longevity) characterized by DECELERATED

aging. At the other extreme there are patients who suffered one or more severe age-related

pathologies in their sixties, seventies and eighties, and died 20-40 years before people of the

same cohort who reached 100 years or more. These patients can be considered as affected by

ACCELERATED aging. In between these two extremes there is a bunch of intermediate

trajectories representing a sort of grey area. Thus, clinically different, classically-defined age-

related diseases such as diabetes, cardiovascular diseases, cancer, dementia, among others,

are indeed the result of a peculiar combination of alterations regarding the same, above-

mentioned shared, limited set of basic mechanisms. Thus, as anticipated by Lazlo Barabasi

some years ago with the conceptualization of “diseasome”, apparently/clinically different

diseases share common mechanisms and genes. According to this integrated view the question

whether aging is a disease in itself becomes meaningless because aging and age-related

diseases are part of a continuum where precise boundaries do not exist. Whether an individual

will follow a trajectory of accelerated or decelerated aging will depend on his/her genetic

background (population genetics) which will interact lifelong with environmental and lifestyle

factors, in a rapidly changing world. Within this scenario it is easy to understand how important

and critical is the search for biomarkers capable of distinguishing between chronological and

biological age and predict/anticipate the rate/trajectory of aging.

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Defining the target group: risk prediction of ageing trajectories

Andrea Maier

Science behind ageing trajectories is rapidly emerging, showing differences in the rate

of ageing already at early ages. This indicates that organ system changes occur even

before diseases become prevalent. Measurements quantifying the ageing process are

not yet defined but highly warranted to disentangle the chronological age from the

biological age, expressing the rate of ageing, in humans.

The EU funded projects PreventIT and PANINI both aim to 1) identify individuals at risk

for future detrimental outcome and 2) tailored approaches to prevent negative

outcomes. Latest evidence on how to measure biological age and its prediction for

ageing trajectories will be presented for different age categories.

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Do biomarkers of ageing reflect real age?

Andreas Simm

Average life span and life expectancy in the industrial nations have grown dramatically

in the last century. Degenerative diseases will become of major interest with age being

an independent risk factor in most of these diseases. Clinicians know that the

chronological age of a patient does not really represent the ‘real’ age of the person.

Biomarkers of ageing should help to characterize the individual biological age and could

be subsequently used to identify individuals at high risk of developing age-associated

diseases or disabilities. A biological age scale can further help to evaluate the success

of preventive actions.

Unfortunately, there are many problems by creating / identifying such biomarkers. We

do not have a gold standard for measuring biological age but do correlate proposed

biomarkers with chronological age. Most of these markers were identified within blood,

which does not reflect all organs.

Skin-autofluorescence related to the accumulation of advanced glycation endproducts is

one of the proposed biomarkers of ageing. On the cellular levels, treatment of

endothelial with AGE inducing dicarbonyls induce cellular senescence. Accumulation of

AGEs in tissue collagen correlate with age and the outcome of patients after cardiac

surgery. Skin autofluorescence reflects tissue AGE modification, correlates with age,

predicts the outcome of patients and can be modified by preventive actions like

exercise. Whereas these data are promising, AGE skin autofluorescence like all other

proposed biological age scales can be used in cohorts only but are unable to monitor an

individual risk.

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Challenges understanding aging through biomarkers across populations: the example of calcium

Tamàs Fülüp

Recent studies have shown contradictory associations between calcium levels and

health outcomes. We suspected these conflicting results were the consequence of more

general issues with how biomarkers are analyzed in epidemiological studies, particularly

in the context of aging. To demonstrate the risks of typical analyses, we used three

longitudinal aging cohort studies and their demographic subsets to analyze how calcium

levels change with age and predict risk of mortality and frailty. We show that calcium

levels either increase or decrease with age depending on the population, and positively

or negatively predict frailty depending on the population; both age and frailty results

showed substantial heterogeneity. Mortality analyses revealed few significant

associations but were likely underpowered. Variation in population composition

(demographics, diseases, diet, etc.) leads to contradictory findings in the literature for

calcium and likely for other biomarkers. Epidemiological studies of biomarkers are

particularly sensitive to population composition both because biomarkers generally have

non-linear and often non-monotonic relationships with other key variables, notably age

and health outcomes, and because there is strong interdependence among biomarkers,

which are integrated into complex regulatory networks. Consequently, most biomarkers

have multiple physiological roles and are implicated in multiple pathologies. We argue

that epidemiological studies of aging using biomarkers must account for these factors,

and suggest methods to do this.

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Motor-Cognitive Interventions to reduce the risk of falls in older adults and neurodegenerative conditions

Anat Mirelman

Gait impairments and falls are ubiquitous among older adults and patients with common

neurological diseases. Normal and safe mobility depends on intact sensory and motor

systems, but there is a growing body of research that specifically links the cognitive sub-

domains of attention and executive function to gait alterations and fall risk. These

domains apparently play a critical role in the regulation of gait especially under

challenging conditions where decisions need to be made in real-time and constant

adaptation is required to manage internal and external factors such as occur in

everyday life situations. Current therapeutic approaches tend to provide interventions

that target a single domain, however, therapeutic interventions, which focus on a

combined motor-cognitive approach, may improve gait and functional everyday mobility

and decrease the risk of falls in older adults and patients with neurological disorders.

Dr. Mirelman will present an overview on the links between motor and cognitive function

and their relationship to mobility, everyday life activity, and falls in ageing. Evidence

from the V-TIME project, a recently completed multi-modal randomized controlled trial,

aimed at improving mobility and fall risk in older adults and patients with Parkinson’s

disease will be presented as an example for a motor-cognitive intervention. The study

compared the effects of a combined treadmill training with virtual reality with a similar

intensity motor training alone on mobility and fall frequency in a diverse group of 302

older adults with a history of falls. The study demonstrated that both interventions were

effective in reducing fall frequency over a 6-month follow up period but the effects of the

treadmill- virtual reality training was significantly larger than that of the treadmill alone.

The presentation will provide a summary of advantages and disadvantages of such an

approach, the fidelity of the method used, the suitability of such intervention for this

population, and the utility of these types of interventions in community settings.

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Age-dependent posttranslational protein modification – driving force in neurodegeneration?

Hans-Ulrich Demuth

The Alzheimer syndrome is the most frequent age-related neurodegenerative disorder which

affects today more than 35million patients world-wide. In Germany alone this number will

exceed 3million people in 2050.

Up to now, there is no medication available which would hold or even reverse the progressive

disease. In recent decades attempts to find disease-modifying drugs have failed. Many of them

have been directed against the deposited Abeta peptides (Aβ), which are product of the

turnover of a membrane protein (APP) of neurons. Some drug approaches where developed to

slow down this protease-driven turnover using small molecule inhibitors, some to immunize

against the Aβ peptides.

However, all of them did not specifically target the very different posttranslational modifications

of Aβ or the Amyloid Precursor Protein (APP). Recent research uncovered among these

modifications very aggregation prone forms which in a prion-like manner induce the formation of

soluble and metastable oligomeric aggregates, which are acknowledged today as the most

neurotoxic agents in the human brain.

Among them, pyroglutamate-3 amyloid-β (pGlu-3 Aβ) is an N-terminally truncated and

pyroglutamate-modified Aβ species which has been shown to be a major component of Aβ

deposited in the plaques and blood vessels of Alzheimer’s disease and Down syndrome brains

(Saido et al., 1995; Lemere et al., 1996).

Formation of pGlu-3 Aβ is a multi-step process whereby the first two N-terminal amino acids,

aspartate and alanine, are cleaved off, exposing glutamate at position 3 on the N terminus of

Abeta. Subsequently, glutamate is post-translationally modified to N-terminal pyroglutamate by

cyclization of the exposed glutamate residue by glutaminyl cyclase and resulting altered

biochemical properties including increased hydrophobicity, high aggregation propensity and

neurotoxicity (Russo et al., 2002; Schilling et al., 2006).

Prevention and therapeutic trials previously conducted in different laboratories showed that

prevention of pGlu-3 Aβ formation by inhibiting the enzyme Glutaminyl Cyclase and pGlu-3 Aβ

mAb immunizations resulted in a reduction in general Aβ and pGlu-3 Aβ pathology (Nussbaum

et al., 2012; Frost et al., 2012). This suggests that presence of pGlu-3 Aβ contributes to

increased plaque burden. Both approaches have recently reached clinical development stage.

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Protein Oxidation and Aggregation in Aging and Senescence

Tilman Grune

Reactive oxygen species (ROS) are formed continuously in the organism even under

physiological conditions. Proteins are prominent targets of various modifications such as

oxidation, glycation, or conjugation with products of lipid peroxidation, leading to the

alteration of their structure and biological function. Under steady state conditions such

modifications are leading to an enhanced degradation of the damaged protein, however,

if the degradation machinery is overwhelmed, protein aggregates are formed.

Such protein aggregates are often randomly formed, are highly reactive, grow with time

and have their own pathophysiological effects which are not yet well understood. These

pathophysiological functions include the formation of reactive species, inhibition of parts

of the ubiquitin-proteasomal system and the induction of lysosomal instability. A large

part of the existing protein aggregates are localized in the autophagosomes of

mammalian cells, but cannot be degraded after fusion with lysosomes, leading to

lysosomal rupture and free protein aggregates, affecting the cellular metabolism. In

order to reduce the effects of free protein aggregates, chaperones assist the formation

of aggresome-like structures which can again be taken up into autophagosomes.

Therefore, protein aggregates underlie a constant fluctuation in the senescent cell and

exert a multitude of effects in the aged cell.

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Proteasome modulation as an anti-aging and anti-aggregation strategy

Niki Chondrogianni

Proteasomes are constituents of the cellular proteolytic networks that maintain protein

homeostasis through regulated proteolysis of normal and abnormal (in any way)

proteins. Proteasome activation in cell lines has been shown to result to cellular lifespan

extension and to exert protein anti-aggregation activity.

Using Caenorhabditis elegans as a model, we analyzed in detail the proteasome status

upon the progression of ageing and Alzheimer's disease (AD) and we investigated the

effects of enhanced proteasome activities on the progression of the above mentioned

phenomena. The obtained results were validated in human and murine cells of neuronal

origin.

Proteasome activation in C. elegans either through genetic means or through

compounds resulted in enhanced levels of proteasome activities that led to a SKN-1-

and proteasome activation-dependent lifespan extension. The elevated proteasome

function conferred lower paralysis rates in various AD nematode models accompanied

by decreased Aβ deposits thus ultimately decelerating the progression of AD

phenotype. More importantly, similar positive results were also delivered in human

neuroblastoma cells and in murine cortical neurons.

Our results suggest that proteasome activation with downstream positive outcomes on

ageing and AD, an aggregation-related disease, is feasible in both a genetic and a non-

genetic manipulation manner in a multicellular organism. Moreover they unveil the need

for identification of anti-ageing and anti-amyloidogenic compounds among the nutrients

found in our normal diet.

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Glycated proteins in nutrition: friend or foe

Katarina Sebekova

During thermal processing of foods, non-enzymatic amino-carbonyl reactions lead to

formation of Maillard reaction products (MRPs) - brownish substances, rendering foods

attractive color, odor and taste. Only a few MRPs are chemically defined yet. In vivo

analogues of MRPs are termed advanced glycation end products (AGEs). AGEs are

formed mainly on long-lived proteins, during ageing, and under conditions of

hyperglycemia, enhanced oxidative and carbonyl stress. AGE-modification alters protein

structure and function. Interaction of AGEs with their receptor RAGE may result in

diabetogenic, inflammatory, or pro-atherogenic responses. Studies in rodents showed

that dietary LMW MRPs are partially absorbed into circulation. Their intake is reflected

by increase in plasma AGEs, their urinary excretion, and accumulation in different

tissues. Dietary MRPs (dMRPs) are the main source of circulating AGEs. Majority, but

not all, studies in rodents and humans showed that diets high in MRPs associate with

negative health effects (e.g. elevated inflammatory markers, impairment in insulin

sensitivity, late complications of diabetes). However, vegetarians, who present more

favorable cardiometabolic risk profile compared with omnivores, consume more dMRPs

and present higher circulating AGE levels. Thus, cause-and-effect relationships

between dMRPs intake and health effects are far from clear by now, as rodents are not

accustomed to ingestion of heated food, thus might not be the ideal model for effects of

dMRPs in humans, and human studies generally use different culinary approach to

prepare high- vs. low-MRP-diets. Formation of MRPs in foods may decrease the

bioavailability of proteins and their susceptibility to gastrointestinal degradation. Other

potentially toxic substances (e.g. acrylamide, aromatic amines) are formed during heat-

treatment of foods. On the other hand, some MRPs may exert antioxidative effects, and

formed melanoidins may exert positive health effects. Recent food habits (e.g.

substitution of breast-milk with infant formulas which contain 100-folds higher amounts

of MRPs; increasing consumption of fast-foods and snacks), and the increase in life

expectancy rates (accompanied by the appearance of novel age-related pathologies, in

which AGEs seem to be implicated) point to the need of further research in this area.

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Cardiac ageing enhances atrial fibrillation or vice versa? Impact of altered miRNA expressional profile and metabolic signalling

for treatment and persistence of atrial fibrillation?

Bernd Niemann

Ageing is said to induce atrial fibrillation (AF) and to reduce therapeutic success during

medical and ablation therapy. Moreover, senescent patients often suffer from perma-nent

AF resulting in the need of oral anticoagulation. Accordingly, elderly patients are at risk for

bleeding complications due to anticoagulation. Therefore, freedom from AF and freedom

from anticoagulation might be an important quality marker for perioperative cardiac surgery

success in aged patients despite indication for anti-arrhythmic therapy is weakened in these

patients following latest guidelines. However, data are lacking that answer the question if

AF is the result of ageing or if AF is causing age-associated atrial changes. Addressing AF

might be as well addressing pathological ageing of the atria and help to improve cardiac

function in patients undergoing cardiac surgery.

Atrial fibrillation from paroxysmal to persistent AF is accompanied by atrial structural

remodeling. Pro-fibrotic signals lead to interstitial fibrosis, which is considered a structural

basis for the development and sustaining of AF and might be understood as to be the

substrate of atrial ageing. Cardiac microRNAs (miRNAs) and adipokines are important

regulators of this interstitial fibrosis. We have characterized patterns of miRNA-signaling

and metabolic signaling (adipokines) in patients suffering from paroxysmal, persistent and

new onset atrial fibrillation.

Clinical and molecular patterns observed in patients with new-onset postoperative AF are

similar to patterns in patients with paroxysmal AF, but are more pronounced in patients with

persistent AF but absent in patients (and elderly patients) with sinus rhythm. Thus,

calendrical age per se might not offer a highly potent substrate and trigger for induction and

persistence of atrial fibrillation, which allows for therapeutic success even in elderly. On the

other hand a pathological environment can perpetuate AF and reduce therapeutic success

in cardiac surgery. Addressing these pathological patterns might offer additional therapeutic

tools to improve therapeutic success and quality of life in aged patients. These senescent

patients might benefit more than younger patients from the opportunity to terminate

anticoagulation and to gain cardiac function due to sinus rhythm.

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Protective effects of caffeine in the cardiovascular system depend on mitochondrial p27 - in aging and disease

Judith Haendeler

Epidemiological studies demonstrate that habitual coffee consumption is associated

with a lower risk for adverse cardiovascular events. We describe a novel mechanism by

which caffeine improves functions of the endothelium and the heart. The effects depend

on p27, known as a cell cycle inhibitor, and moreover, on its caffeine-induced

translocation to mitochondria. Only mitochondrial p27 phosphorylated on two specific

residues enhances migration of endothelial cells. Furthermore, it protects

cardiomyocytes against apoptosis. p27-deficiency and aging decrease respiration in

heart mitochondria and the latter is rescued by caffeine, which induces transcriptome

changes in a p27-dependent manner affecting mostly genes relevant for mitochondrial

processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic

mice, and increases mitochondrial localization of p27. Moreover, p27 is necessary for

myofibroblast differentiation, and thus, proper infarct healing. Our data directly link the

protective effects of caffeine in the cardiovascular system to mitochondrial p27.

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Oral anticoagulation and polypharmacy in older adults with atrial fibrillation

Markus Gosch

Atrial fibrillation is the most frequent cardiac arrhythmia. The prevalence is around 1 to 2

% and increases with age up to 5 to 15 % in people aged over 80 years. It is one of the

strongest risk factors for stroke. In case of atrial fibrillation oral anticoagulation is a well

known treatment strategy that leads on one hand to a significant reduction of strokes in

these patients, on the other hand to a higher risk of bleeding. Regarding the guidelines

the indication of oral anticoagulation treatment in patients with atrial fibrillation seems to

be quite easy. However, older and very old patients are underrepresented in clinical

trials and we get only some information about this group of patients in a very short

chapter called „special situations“. Many questions in the management of older and very

old patients remain unanswered. It is not only the question about anticoagulation itself,

since few years it is more a discussion on the choice of treatment. Beside vitamin K-

antagonists, four different DOAC’s are available. Regarding the recommendations of the

guidelines DOAC‘s should be preferred compared to Vitamin K antagonists.

As mentioned in the guidelines we need special considerations for older and very old

adults. Multimorbidity, polypharmacy and geriatric syndroms have a high prevalence in

this age group and an enormous impact on management of oral anticoagulation.

Patients above 80 has a significant risk of bleeding compared to younger age groups,

but even this age group benefits from oral anticoagulation. What we need is an

individual approach based on guidelines, current literature and clinical assessment.

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Gender related aspects of the cardiovascular system and its impact on progression and aging.

Sandra Eifert

Mortality rates in cardiovascular disease differ highly between men and women,

independent of treatment options. Applying the gender lens to cardiovascular aging is of

major clinical interest. That accounts for the entire course of disease; from risk factors

via onset to therapeutic options. They may play a leading role in cardiovascular disease

progression and aging. Cardiovascular risk factors including renal failure, and hormonal

influences were reviewed and their gender-specific distribution isreported on.

The levels of sex hormones decrease with age and thus, may potentially contribute to

cardiovascular aging and the adaption to certain physiologic or non physiologic

conditions. Sex-specific factors implicating endothelial function/ dysfunction will be

recognized because of its clinical relevance to preventive and therapeutic strategies.

Outcomes of men and women in relation to age will be reported in aortic disease, aortic

stenosis as well as heart failure.

Female gender has become an independent risk factor of survival after the majority of

cardiosurgical procedures. Severely impaired left ventricular ejection fraction

independently predicts survival in men, whereas age does in females.

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Historical Overview of Research on Lifespan Extension and its Ethical Implications

George M. Martin

The human aspiration to greatly enhance one’s lifespan probably dates to the early history of Homo

sapiens and the evolution of his neocortex. Recent research on the comparative genomics of our

species and that of our closest cousin, the chimpanzee, in fact points to the emergence of specific

genetic loci responsible for our extensive six-layered cerebral cortex (KS Pollard Nature, 2006). The

idea that scientific research might bring these wishes to fruition might be dated to the 16th century

when, for example, individuals such as Robert Boyle (1627–1691), a founding member of the Royal

Society, is said to have proposed experiments as "to replace the blood of the old with the blood of

the young" (https://en.wikipedia.org/wiki/Life_extension ) – a remarkably contemporary enterprise

(https://www.technologyreview.com/s/603242/questionable-young-blood-transfusions-offered-in-us-

as-anti-aging-remedy/ )!

Most contemporary geroscientists, however, would probably point to the landmark dietary restriction

experiments on rats as the beginning of the modern era of lifespan extension research (C.M McCay,

et al. Scientific Monthly, 1934). Numerous species have since been shown to respond to such

interventions, although much more research is needed to establish dose-response curves as

functions of genetic backgrounds.

A huge volume of basic research has finally led to what may well be the first US Food and Drug

Agency approved clinical trial of an agent (Metformin) for the enhancement of lifespan

(https://www.wired.com/story/this-pill-promises-to-extend-life-for-a-nickel-a-pop). Meanwhile, a range

of substances are being consumed without such clinical trials because of their marketing as

Nutraceuticals. Ethical concerns about efficacy and about possible untoward effects in genetically

susceptible individuals who purchase “anti-aging” products need to be addressed in such cases,

however. The sale of hormones for such treatments was estimated to yield circa $50 billion in sales

back in 2009 (http://articles.chicagotribune.co2009-06-15/news/0906140132_1_anti-aging-

hormones-ama-council).

The major concern for legitimate research-based interventions to extend lifespan is the paucity of

evidence that such interventions would increase the ratios of “healthspan/lifespan” in most

individuals. One could imagine, for example, that an intervention that led to enhanced physiological

robustness and resilience might extend the duration of a lethal illness and thus decrease that ratio

(M. Kaeberlein et al., Science, 2015).

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Rejuvenation biotechnology: why age may soon no longer mean aging

Aubrey de Grey

People are living longer than they used to - no longer because of reductions in child

mortality, but because we are postponing the ill-health of old age. But you’ve seen

nothing yet: regenerative medicine and other new biomedical technologies will

eventually be so comprehensive that people will stay truly youthful however long they

live - which means they may mostly live very long indeed. The social and economic

consequences of this transition will pervade every aspect of our lives. I will discuss

recent progress towards the implementation of this comprehensive “rejuvenation

biotechnology”.

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The Shape of Lives to Come

Ian Richard Ground

The ethical challenges posed by clinical and technological advances in the

understanding of ageing are the subject of welcome controversy across political and

social, economic and environmental dimensions.

But ethics is not concerned not just with the good or bad, utopian or dystopian

consequences of our decisions. In a world facing all too many challenges, such

utilitarian calculations have too many variables to offer much certainty.

A rich philosophical tradition holds that Ethics also concerns our conception of what it is

to lead a flourishing – as the Greeks held – eudaemonic human life. A central idea is

that how we lead our life – our commitments, life decisions and so on - is partly

constituted by our conception of the shape of that life.

In my contribution to the panel discussion, I wish to highlight the ethical challenges

posed by the advances in understanding ageing to our conception of the shape of our

lives and what it means for such lives to flourish. If we come to think of ageing as a

disease, what are the consequences for the rest of our ethical and conceptual scheme?

How we reconcile that radical conceptual change in our thinking about ageing - that

many would have us enthusiastically embrace - with what may be a deeper truth: that it

is not merely our bodies, but we who age?

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.

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Ethical questions of biogerontological research. An overview of the debate

Zóe Rheinsberg

Significant advances have been achieved in the field of biological ageing research.

They give raise to hopes in the context of challenges related to population ageing but

also to important ethical questions. These questions need to be addressed both by

scientific research and debate and by a broad discourse involving the general public.

The following sectors should be explored and discussed: (1) ethical, legal, and social

aspects of biogerontological research, and its methods and goals, (2) specific

challenges in research ethics, and (3) specific ethical implications for physicians. It is

crucial that the debate focuses on realistic scenarios of what could be achieved by

biogerontological research in closer future. A postponement of the onset of age-related

diseases and a moderate extension of the human lifespan seem to be a more suitable

basis for discussion than a radical life extension and the entire abolition of age-related

disease and suffering.

Concrete questions which need to be addressed relate to the necessity and dimension

of funding for biogerontological research, the accessibility of potential medical

applications, and the influence of biogerontological research on perception and images

of age, ageing, and old persons. While this is already happening in the scientific

community the public dialogue is still at its beginnings. Discourse projects can be one

approach to initiate and inform a public debate on ethical, legal, and social questions of

biogerontological research.

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Epigenetic stress response and stem cell aging

Lenhard Rudolph

Adult tissue stem cells contribute to the lifelong maintenance of 0rgan homeostasis and

regeneration. However, the functionality of stem cells declines during aging and there is

emerging evidence for the clonal dominance of mutant stem cells. Both processes

contribute to the evolution of aging associated dysfunctions and diseases but molecular

mechanisms that impair the function of stem cells during aging remain incompletely

understood. Our recent work revealed that alterations in epigenetic stress responses

lead to an aberrant activation of developmental pathways that impair the self renewal

and regenerative capacity of muscle stem cells. Recent studies from the laboratory of

David Scadden indicate that heterogeneity of epigenetic memory in hematopoietic stem

cell is linked to the number of cell divisions and determines the heterogeneity in the

functionality of HSCs during aging. Interestingly, the vast majority of gene mutations

leading to clonal dominance of HSCs during aging affect epigenetic regulators.

Together, these data indicate that alteration in epigenetic memory and stress responses

represent driving forces for the decline of stem cell function and the selection of stem

cell mutation during aging. During my talk I will present new data on physiological

conditions and molecular mechanisms that may contribute to alterations of the

epigenome and gene regulation in aging stem cells and thus to disease development in

aging.

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Understanding muscle stem cell regenerative decline with aging

Pura Munoz-Canove

Skeletal muscle has a remarkable capacity to regenerate by virtue of its resident Pax7-

expressing stem cells (satellite cells), which are normally quiescent in the adult. Upon

injury, quiescent satellite cells activate and proliferate, to subsequently differentiate and

form new myofibers or self-renew to restore the quiescent satellite cell pool. We have

shown that these long-lived resting stem cells attenuate proteotoxicity and avoid

senescence through basal autophagy. This protective “clean-up” system is lost during

aging, resulting in stem cell regenerative decline. We therefore propose that loss of

proteostasis is causally implicated in muscle stem cell aging, a process that can be

targeted for rejuvenation.

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Bioengineered matrices to regenerate the heart – The Zurich LifeMatrix Programm

Maximilian Emmert

Cardiovascular disease represents a major cause of morbidity and mortality worldwide

accounting for the death of numerous patients. The concept of regenerative medicine

comprising cell-based therapies, bio engineering technologies and hybrid solutions has

been suggested as a next generation approach for affected patients. In particular,

cardiovascular tissue engineering has been demonstrated to be a promising concept to

generate living, autologous cardiovascular structures with the ability to remodel and to

grow which may be particularly beneficial for children. However, although these

regenerative concepts have shown great potential in experimental studies, the safe and

effective translation into a widespread clinical setting is still limited. Furthermore, the

currently utilized ``classical`` approaches are still too complex with regards to logistical

and financial effort. Therefore, significant simplification is needed and in particular, so

called off-the-shelf concepts may represent an attractive option to overcome the current

problems towards clinical translation.

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Mesenchymal Stem Cells-on-Chips/Organ-on-Chips/Chips in Mesenchymal Organs – Monitoring Healing Kinetics:

an ageing perspective

Günter Lepperdinger

Stem cells play an important role during development and regeneration, and their

dysfunction is associated with a variety of diseases, such as sarcopenia, osteopenia, or

several classes of cancer. Activated stem cells bring forth specific progenitor cells,

which promote tissue remodeling, repair and regeneration (RRR). Adult human

mesenchymal stem cells (MSC) can be readily isolated from different sources, and

many clinical trials have been successfully performed previously. Consequently, many

therapies recapitulating RRR are currently being implemented in clinical settings. Major

tissue changes during RRR are constituted through the deposition of material into the

pericellular space, be it soft extracellular matrix (ECM) or dense load-bearing

substances such as collagenous hydroxyapatite. In vivo quantitative analyses thereof

are still demanding. We first developed impedance microsensors for in vitro use, which

allowed to assess cell attachment and viability as well as bipotential differentiation

capacity, thus proofing that impedance changes enables documentation of MSC quality

in a fast, efficient and reliable fashion. Based on this biochip technology we have

developed implantable titanium dioxide-coated bioimpedance sensors. After

implantation, the sensory system revealed in subcritical defects of rabbit calvaria distinct

differences between granulation and soft tissue formation within two weeks, thus

allowing the accurate assessment of bone healing prior the formation of a cortical bone

layer. Hence this technology enables diagnosis of early healing deficiencies or

decreasing implant stability. Accounting for that specific early information allows

commencing with and adjustment of appropriate medicinal interventions.

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Effects of pharmacological inhibition of aging-associated pathways on Drosophila lifespan

Alexey Moskalwev

Mutations of the PI3K, TOR, iNOS, and NF-κB genes increase lifespan of model

organisms and reduce the risk of some aging-associated diseases. We studied the

effects of inhibitors of PI3K (wortmannin), TOR (rapamycin), iNOS (1400W), NF-κB

(pyrrolidin dithiocarbamate and QNZ), and the combined effects of inhibitors: PI3K

(wortmannin) and TOR (rapamycin), NF-κB (pyrrolidin dithiocarbamates) and PI3K

(wortmannin), NF-κB (pyrrolidine dithiocarbamates) and TOR (rapamycin) on

Drosophila melanogaster lifespan and quality of life (locomotor activity and fertility). Our

data demonstrate that pharmacological inhibition of PI3K, TOR, NF-κB, and iNOS

increases lifespan of Drosophila without decreasing quality of life. The greatest lifespan

expanding effect was achieved by a combination of rapamycin (5 μM) and wortmannin

(5 μM) (by 23.4%). The treatment of Drosophila melanogaster with 10 non-steroidal

anti-inflammatory drugs (NSAIDs: CAY10404, aspirin, APHS, SC-560, NS-398, SC-

58125, valeroyl salicylate, trans-resveratrol, valdecoxib, licofelone) leads to extension of

lifespan, delays age-dependent decline of locomotor activity and increases stress

resistance. The lifespan extending effects of APHS, SC-58125, valeroyl salicylate,

trans-resveratrol, valdecoxib, and licofelone were more pronounced in males,

valdecoxib and aspirin - in females. We demonstrated that lifespan extension effect of

NSAIDs was abolished in flies with defective genes involved in Pkh2-ypk1-lem3-tat2

pathway.

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The Path towards Translating Senolytic Drugs and Other Interventions Targeting Aging Processes into Clinical Treatments

James Kirkland

Interventions targeted at fundamental mechanisms of aging hold promise for enhancing

healthspan by delaying, preventing, or alleviating a range of age-related diseases and

conditions—a theory termed the “geroscience hypothesis.” Among the more promising

interventions are senolytic agents, drugs that selectively eliminate senescent cells.

Early, proof-of-concept Phase 2 clinical trials will be a critical step in translating these

agents and others emerging from cell culture and animal preclinical studies into clinical

practice. The goals of such proof-of-concept trials for newer interventions include

generating preliminary signals of efficacy in age-related diseases or outcomes that will

test the value of proceeding to larger Phase 3 trials. Drugs that target aging processes,

such as metformin, for which there are already extensive clinical data related to other

indications, are at the point of being tested in Phase 3 trials, such as the proposed

TAME study. In addition to generating data about effectiveness, such trials need to be

designed to contribute data and biological samples to support basic research through

strategic networks, as well as furthering dialogue with regulatory agencies about

registration indications. Frameworks for proof-of-concept and later stage trials that

target age-related chronic diseases, geriatric syndromes, or resilience to stressors will

be considered. We propose that a strategic infrastructure and shared resources are

needed to accelerate translation of therapies targeting fundamental aging processes

into clinical treatments.

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Cellular senescence and aging

Judith Campisi

Cellular senescence is a complex cell fate decision that entails an essentially

irreversible arrest of cell proliferation coupled to a multi-component secretory phenotype

(termed the senescence-associated secretory phenotype or SASP). The senescence

response, and particularly the SASP, is most likely an evolutionary trade-off. That is, it

likely evolved to benefit young organisms by promoting tissue repair and remodeling,

but becomes maladaptive and compromising to tissue homeostasis later in life. This

trade-off arises because the SASP includes a variety of pro-inflammatory cytokines,

chemokines, growth factors, proteases and bioactive lipids, many of which can cause or

contribute to local and systemic inflammation – a hallmark of aging. Several lines of

recent evidence, primarily from mouse models, suggest that the selective clearance of

senescent cells from aged individuals can ameliorate a variety of age-related

phenotypes and pathologies. The challenge for applications to humans will be to

preserve the beneficial effects of senescent cells, while eliminating their deleterious

effects later in life.

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DNA damage: impact on aging, neurodegeneration and the effect of nutritional interventions

Jan Hoeijmakers

The molecular basis of aging (-related diseases) is one of the main unsolved questions in

biology. Aging appears remarkably plastic: e.g. suppressing insulin signalling extends lifespan

in worms, flies and mice. On the other hand, virtually all premature aging syndromes in man

provide links with genome instability. We have generated mouse models which strikingly mimic

human DNA repair deficiency disorders and display wide-spread accelerated aging, revealing a

strong link between unrepaired DNA damage and many features of aging. E.g. Ercc1∆/- mice

defective in ≥3 repair pathways show extensive premature multi-morbidity in post-mitotic and

proliferative tissues limiting lifespan to 4-6 month. Simultaneously these mice exhibit an anti-

aging ‘survival response’, which suppresses growth and enhances maintenance, resembling the

longevity response induced by dietary restriction (DR). Interestingly, subjecting these progeroid,

dwarf mutants to actual (30%) DR tripled remaining lifespan, and drastically retarded numerous

aspects of accelerated aging, with the neuronal system benefitting disproportionally. E.g. DR

animals retained 50% more neurons and maintained full motoric function, delaying motor

decline >20(!)-fold. Repair-deficient Xpg-/- mice also showing many premature aging symptoms

responded similarly. The DR response in Ercc1∆/- mice resembled wt DR including (further)

reduced insulin signaling. Interestingly, ad libitum Ercc1∆/- liver expression profiles showed

declining expression of long genes, consistent with genome-wide accumulation of stochastic,

transcription-blocking lesions, which affect long genes more than short ones. Similar findings

were made in human brain profiles upon aging, demonstrating relevance for normal aging in

humans. DR in repair-deficient mice alleviated this decline, indicating that DR prolongs genome

function. We will present phenotypes of conditional DNA repair models targeting aging to

selected organs and connections with the unfolded protein response and proteinopathies

(Alzheimer’s and Parkinson diseases). Our findings strengthen the link between DNA damage

and aging, establish Ercc1∆/- mice as powerful model for identifying interventions to promote

healthy aging, reveal untapped potential for reducing endogenous damage, provide new venues

for understanding the molecular mechanism of DR, and suggest a counterintuitive DR-like

therapy for human progeroid genome instability syndromes and DR-like interventions for

preventing neurodegenerative diseases.

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Posters (in alphabetical order)

The Poster Award

Ceremony

Sunday 3rd

of September from 11:30 to 11:45

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(1) Carbon nanoparticles cause loss of gap junctional intercellular

communication in the lung and the endothelium

Nilo Ale-Agha1, Tim Spannbrucker1,2, Klaus Unfried2, Yogi Altschmied1, Jojo Haendeler1

1Heisenberg group - Environmentally induced Cardiovascular Degeneration, Clinical Chemistry, University of

Duesseldorf, and IUF-Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany, 2Environmentally induced skin and lung aging, IUF-Leibniz Research Institute for Environmental Medicine,

Duesseldorf

Introduction: Gap junctional intercellular communication (GJIC) pathways are involved in a

variety of vascular and pulmonary functions. Gap junctions composed of connexin (Cx)

molecules are clusters of intercellular channels connecting adjacent cells. The loss of Connexin

43 (Cx43), one of the major connexins in the vascular and pulmonary systems, and GJIC was

suggested to be related to senescence and many cardiovascular and pulmonary disorders. It is

known that environmental factors like industrial or consumer derived pollution affect the

cardiovascular and pulmonary systems. Senescent endothelial cells are associated with

cardiovascular diseases, and lung cell senescence is supposed to impair lung function. We

have shown that non-cytotoxic, non-inflammatory concentrations of carbon nanoparticles (CNP)

induce senescence both in human primary endothelial cells (EC) and rat lung epithelial cells

(RLE).

Objective: The aim of this study was to investigate whether CNP affect GJIC in the lung and

endothelium and might therefore be involved in cardiovascular and pulmonary aging processes.

Material and Methods: CNP particle suspensions were applied to EC, RLE or C57Bl/6 mice

(pharyngeal aspiration). GJIC was measured by dye transfer assay. The localization of Cx43

was assessed by immunofluorescence staining in lung sections. Phosphorylation and protein

levels of Cx43 were determined by immunoblot.

Results: Two weeks incubation of cells with non-toxic CNP concentrations showed increased

levels of senescence-associated beta-galactosidase and the cell cycle inhibitor p21, two known

markers for cellular aging. The same concentration of CNP resulted in a dramatic reduction of

GJIC in both cell types, which was due to a significant change in phosphorylation and

internalization of Cx43. Thus, CNP-induced senescence is accompanied by loss of GJIC. CNP

applied in non-inflammatory concentrations into mice in vivo reduced Cx43 protein levels, while

the phosphorylation of Cx43 was upregulated in the lungs of CNP treated mice.

Conclusion: These data indicate that Cx43, which is essential for endothelial and pulmonary

functionality, is affected by CNP and that loss of GJIC is involved in CNP-induced senescence.

Further studies have to clarify whether a causal link between CNP-induced senescence and

loss of GJIC exists.

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(2) Protective functions of mitochondrial TERT in age-associated

cardiovascular diseases

Yogi Altschmied, Christine Goy, Mark Zurek, Florian von Ameln, Niloofar Ale-Agha, Jojo Haendeler

Heisenberg group - Environmentally induced Cardiovascular Degeneration, Clinical Chemistry, University of Duesseldorf, and IUF-Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany

Introduction: Age-associated cardiovascular disorders are associated with mitochondrial

dysfunction, which causes reduced respiratory chain activity, decreased mitochondrial ATP

production, an increase in reactive oxygen species (ROS) and cell death. We have previously shown

that the catalytic subunit of telomerase, Telomerase Reverse Transcriptase (TERT) is not only

present in the nucleus, but also in mitochondria, where it binds to mtDNA and protects it from

damage. Furthermore, TERT improves mitochondrial respiration and reduces mitochondrial ROS

formation and both these functions can presumably ascribed to mitochondrial, rather than nuclear

TERT.

Objective: The aim of this study was to characterize the functions of mitochondrial TERT in age-

associated cardiovascular diseases ex vivo and in vivo.

Materials and Methods: To analyze specifically the role of mitochondrial TERT we transduced

different cardiovascular cells with lentiviral expression vectors for mitochondrially-targeted TERT.

Furthermore, we created a unique new mouse model, in which the animals contain TERT exclusively

in the mitochondria (mitoTERT mice). Apoptosis as well as cytosolic and mitochondrial ROS were

measured by flow cytometry and ATP with a luminometric assay. Respiratory chain activity was

measured by oxygraphy. Myofibroblast differentiation of isolated cardiac fibroblasts was assessed

measuring specific markers by immunoblotting and semi-quantitative real-time PCR. Experimental

myocardial infarction was set using ligation of the left ascending coronary artery in an open chest

model.

Results: Mitochondrial TERT showed protective functions in different cells of the cardiovascular

system, as it specifically reduced mitochondrial ROS levels, protected endothelial cells and

cardiomyocytes against apoptosis. Moreover, mitochondrial TERT was sufficient to compensate for

TERT-deficiency in mitochondrial respiration and ATP production in the heart as well as in disturbed

cardiac myofibroblast differentiation. In addition, while TERT-deficient animals showed worse

outcome after myocardial infarction compared to wildtype littermates, mitochondrial TERT was

sufficient to rescue this defect.

Conclusion: Our data show for the first time that mitochondrial TERT is required and sufficient to

protect cells of the cardiovascular system ex vivo and in vivo against damage and loss of

functionality commonly observed in age-associated cardiovascular diseases and complications, like

e.g. atherosclerosis and myocardial infarction.

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(3) Human HSCs show changes in frequency, polarity and cell

division kinetics upon aging

Amanda Amoah, Anja Keller, Ramiz Emini, Markus Hönicka, Andreas Liebold, Carolina Florian, Hartmut Geiger.

Hematopoietic stem cells (HSC) are crucial for maintaining blood homeostasis

throughout the life of an organism. Recent findings using mouse models, however,

show distinct functional and phenotypic changes in HSCs upon aging, which are

reversible. To better explore the prospects of rejuvenation, we sought to characterize

aging-induced changes in human HSCs. Our results show that although the number of

total bone marrow (BM) cells remain unchanged, the number of multipotent

hematopoietic stem and progenitor cells (HSPC) (CD34+CD38-) and the number of

HSCs (CD34+CD38-CD90+) increase upon aging. Furthermore, we observed a delay in

aged HSCs in the response to cytokine stimulation ex vivo. Similar to murine HSCs,

aged human HSCs show a significant decrease in the frequency of cells that present

with a polar distribution of tubulin and the small RhoGTPase Cdc42 in the cytoplasm

and Ac-H4K16 in the nucleus. Collectively these findings identify functional and

phenotypic changes occurring in human HSCs upon aging.

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(4) Dietary flavanol monomer (-) epicatechin enhances endothelial

function - causal role of SNAP23?

Karin Aufenvenne1, Christine Goy1, Christian Heiss2, Yogi Altschmied1, Jojo Haendeler1

1Heisenberg group - Environmentally induced Cardiovascular Degeneration, Clinical Chemistry, University of

Duesseldorf, and IUF-Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany, 2Division of Cardiology, Pulmonology, and Vascular Medicine, University of Duesseldorf

Introduction: Diet is a major determinant of health as well as healthspan. Cocoa

flavanols have emerged as important dietary bioactives that may be able to improve

cardiovascular health. However, the mechanisms of action are not established.

Objectives: In the present work, we aimed at identifying molecules activated by

flavanol related bioactives, e.g. (-) epicatechin using gene expression analyses and

confirmed the findings with endothelial cell models. We hypothesized that regular

consumption of plant flavanols may lead to improved endothelial function.

Material and Methods: Microarray based analyses of whole blood samples from

healthy volunteers. Hypothesis driven analyses of gene expression results; analysis of

SNAP23 by immunoblot; measurements of NO bioavailabilty and eNOS phosphorylation

were performed. Transient transfections as well as lentiviral transductions of endothelial

cells with expression vectors for SNAP23 were performed.

Results: We first performed microarray based expression analyses of whole blood

samples from healthy volunteers randomly and double blinded consuming cocoa

flavanols (450 mg bi-daily) or placebo over one month. In parallel with improved

vascular function, the analyses showed a striking upregulation of approximately 500

transcripts. One of the most prominent upregulated genes by flavanols is SNAP23. To

investigate a causal relationship ex vivo, we first investigated whether (-) epicatechin

increases SNAP23 protein levels. Indeed, SNAP23 was increased up to 3-fold. Along

the same line eNOS was activated and NO bioavailability was increased by (-)

epicatechin. Moreover, overexpression of SNAP23 in combination with (-) epicatechin

did not enhance eNOS phosphorylation further, suggesting the same pathway.

Conclusion: Flavanols increase flow-mediated dilation. This effect depends on NO and

on SNAP23 as demonstrated in endothelial cells.

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(5) Novel Amide AGEs as Oxidative Stress Markers

T. Baldensperger, Halle (Saale)/D, Prof. Dr. M. A. Glomb, Halle (Saale)/D

Prof. Dr. M. A. Glomb, MLU Halle-Wittenberg, Institute of chemistry – Food chemistry, Kurt-Mothes Straße 2, 06120 Halle (Saale), D. [email protected]

The demographic change in Germany and improving medical care lead to progressive

ageing of our society. As a result an increase of age associated diseases like diabetes,

atherosclerosis and cancer is expected. In order to face these future problems a

detailed knowledge of molecular mechanisms leading to the decline of organ functions

and tissue homeostasis is crucial. There is rising evidence that posttranslational

modifications of proteins play a key role in ageing processes. One major aspect of

posttranslational modification in vivo is the enzymatic acylation of proteins. Recent

publications indicate that Maillard processes and fat metabolism also lead to

accelerated acylation of proteins. This means fundamentally important enzymatic

processes are paralleled by non-enzymatic pathways. The physiological importance and

correlation to ageing of such non-enzymatic acylation pathways is not understood yet.

In order to analyze non-enzymatic acylation a highly sensitive HPLC-MS/MS method

was developed. Authentic reference standards of the novel amide advanced glycation

endproducts (AGEs) N-glyoxylyl lysine and N-pyruvoyl lysine were synthesized. First

screenings revealed N-glyoxylyl lysine as a glyoxal and N-pyruvoyl lysine as a

methylglyoxal specific AGE. N-glyoxylyl lysine was established as a further product of

the N-carboxymethyl lysine (CML) reaction cascade and N-pyruvoyl lysine as a further

product of the N-carboxyethyl lysine (CEL) reaction cascade. Consequently we

proposed a mechanism including isomerization and oxidation of the central intermediate

of the CML/CEL reaction cascade in analogy to the oxidation step established for the

formation of carboxylic acids in the well-known Strecker degradation. In fact,

N-glyoxylyl lysine and N-pyruvoyl lysine were formed exclusively under aerated

conditions. In addition, artifact amide formation directly from glyoxylic acid, pyruvic acid

or from aldol condensation products were ruled out. Due to the strict correlation of

N-glyoxylyl lysine and N-pyruvoyl lysine formation to oxidative conditions we aim to

establish these novel amide AGEs as marker structures for oxidative stress in vivo.

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(6) Impact of advanced glycation end products on macrophage activation.

Veronika Bezold1, Rüdiger Horstkorte1, Toni Ehrhardt2, Matthias Jung2, Kaya Bork1

1Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany

2Department of Psychiatry, Psychotherapy and Psychosomatics, Martin-Luther-University Halle-Wittenberg, Halle

(Saale), Germany

Advanced glycation end products (AGEs) and their receptor have been implicated in the

progressions of many diseases, such as diabetes and atherosclerosis, and are also

critical for pathologic changes in chronic degenerative diseases, such as Alzheimer's

disease and Parkinson's disease. AGEs increase with age and have been investigated

as a marker for aging. It is discussed if age-associated changes in immune system

development and function are related to an increased level of AGEs [1].

Differentiation of monocytes into macrophages is a key step in innate immune

response. The human monocytic cell line THP-1 can easily be differentiated into

macrophages using various chemical stimuli. Therefore this cell line is often used as a

model for differentiation studies. We treated undifferentiated and differentiated THP-1

cells with methylglyoxal (MGO) to increase the formation of AGEs and analyze the

activation of the cells. We could show that the increased formation of AGEs influences

the expression of the activation marker CD14, interleukin 1-beta, interleukin 6,

interleukin 8, interleukin 10 and TNF-alpha. In addition, we analyzed the expression of

the AGE-receptor RAGE after treatment with MGO. RAGE is a transmembrane receptor

expressed in various immune cells. The interaction between RAGE and its ligands is not

clearly understood regarding cellular signaling, though it is thought to result in pro

inflammatory gene activation [2].

References [1] G. Glorieux, R. Vanholder, N. Lameire, Eur. J. Clin. Invest. 2011, 12, 1015-1018.

[2] A. Bierhaus, S. Schiekofer, M. Schwaninger, M. Andrassy, P.M. Humpert, J. Chen, M. Hong, T. Luther, T. Henle, I. Klöting, M. Morcos, M. A. Hofmann, H. Tritschler, B. Weigle, M. Kasper, M. A. Smith, G. Perry, A.M. Schmidt, D. M.Stern, H.U.Häring, E. Schleicher, P.P. Nawroth, Diabetes 2001, 50, 2792-2809.

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(7) Development of an age-specific genome-scale model of skeletal

muscle metabolism

Andrea Cabbia, MSc

Biomedical Engineering Eindhoven University of Technology (NL)

Skeletal myocytes are among the most metabolically active cell types, implicated in

nutrient balance, contributing to the insulin-stimulated clearance of glucose from the

blood, and secreting myokines that contribute in regulating inflammation and the ageing

process. The loss of muscle mass and strength with age (sarcopenia), is a risk factor for

cardiovascular and metabolic diseases, it increases the risk of falls, of developing frailty

and disabilities, and results in an impairment in the quality of life and autonomy of an

individual.

An active lifestyle is the most immediate and accessible treatment to prevent

sarcopenia, with a considerable impact on the ageing process: PANINI is a European

Training Network whose aim is understanding how lifestyle factors can influence healthy

ageing. In this context, we present the first age-specific genome-scale metabolic model

of the skeletal muscle, a mathematical representation of the myocyte metabolic network

in the elderly, built using RECON2, the human metabolic reconstruction, and gene

expression data, gathered from older adults' muscle tissue biopsies.

This model will be used to analyze patient-specific data for potential mechanisms able

to explain the different ageing paces of different individuals and to investigate the

effectiveness of different nutritional and physical exercise regimes in stimulating post-

exercise protein synthesis, which is often impaired in the elderly.

The aim is to identify an optimal and personalized lifestyle change intervention able to

prevent the onset of sarcopenia.

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(8) Involvement of mitophagy in the elimination of damaged

mitochondria during the process of UVB-induced senescence

Maria Cavinato1, Rafal Koziel1, Sophia Wedel1, Brigitte Jenewein1, Martin Hermann2, Nikolaus Romani3, Pidder Jansen-Dürr1

1Institute for Biomedical Aging Research, Universität Innsbruck, Innsbruck, Austria

2Department of Anesthesiology and Critical Care Medicine, Medical University of Innsbruck, Innsbruck, Austria

3Department of Dermatology and Venerology, Medical University of Innsbruck, Innsbruck, Austria

Skin aging is the result of two types of aging, “intrinsic aging” an inevitable consequence

of physiologic and genetic determined changes and “extrinsic aging” which is dependent

on external factors like exposure to sunlight, smoking, dietary habits among others. UV

and other forms of ionizing radiation cause skin injury through the generation of free

radicals and other oxidative byproducts as well as DNA damage. The oxidized proteins

are generally degraded by the ubiquitin-proteasome system or by autophagy, and

alterations on these pathways lead to accumulation of damaged molecules. The activity

of proteasomes and autophagic organelles is regulated by overlapping signals, and

regulatory cross-talk between both quality control systems has been described.

Likewise, excessive ROS production leads to impairment of mitochondria with

consequences that are related to several age-related diseases as well as to the

physiology of normal aging. We have previously demonstrated that inhibition of

proteasomal degradation of damaged proteins and activation of autophagosome

formation are early events in UVB-induced senescence of human dermal fibroblasts

(HDF), dependent on UVB-induced accumulation of reactive oxygen species (ROS).

Here we show that UVB treatment of HDFs leads to impaired mitochondrial function,

damage to mitochondrial structure and disruption of mitochondrial network and that

these damaged organelles are eliminated by mitophagy. Under these conditions,

mitophagy receptor Bnip3L/Nix is differentially regulated in fibroblasts. Elimination of

Bnip3L/Nix increases cell proliferation and inhibits mitophagy activation upon UVB

treatment. These findings have potential implications for fundamental as well as

translational research into skin aging, and in particular photoaging.

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(9) Investigating the relationship between protein glycation and

aggregation in cells and tissues

Simone Di Sanzo

1. Ori, A. et al. Integrated Transcriptome and Proteome Analyses Reveal Organ-Specific Proteome Deterioration in Old Rats. Cell Syst. 1, 224–237 (2015).

2. Salahuddin, P., Rabbani, G. & Khan, R. The role of advanced glycation end products in various types of neurodegenerative disease: a therapeutic approach. Cell. Mol. Biol. Lett. 19, (2014). 3. Vlassara, H. & Uribarri, J. Advanced glycation end products (AGE) and diabetes: cause, effect, or both? Curr. Diab. Rep. 14, 453 (2014).

4. Stinghen, A. E. M., Massy, Z. A., Vlassara, H., Striker, G. E. & Boullier, A. Uremic Toxicity of Advanced Glycation End Products in CKD. J. Am. Soc. Nephrol. 27, 354–370 (2016).

5. Snow, L. M., Fugere, N. A. & Thompson, L. V. Advanced Glycation End-Product Accumulation and Associated Protein Modification in Type II Skeletal Muscle With Aging. Journals Gerontol. Ser. A Biol. Sci. Med. Sci. 62, 1204–

1210 (2007).

The homeostasis of the proteome of cells is required to maintain the function of organs

and it was shown to decline during aging. The proteostasis is influenced by changes in

protein localization, protein abundance and post-translational modification1. The

formation of advanced glycation end products (AGEs) is a non-enzymatic

posttranslational modification that has been shown to influence the activity and

aggregation propensity of both extracellular and intracellular proteins. AGEs are

associated with oxidative stress and inflammation, processes that lead to cardiovascular

disease, diabetes, chronic kidney disease (CKD), and neurodegenerative diseases2–4.

Although a handful of specific AGE-modified proteins have been identified for example

in aging skeletal muscle5, a detailed characterization of the targets of AGEs and the

relationship between this modification and protein aggregation are currently missing. In

this project, we aim to identify the preferential protein targets of AGEs in cells and

tissues. For this purpose, we develop a proteomic workflow based on selective

enrichment of AGE-modified proteins coupled to mass spectrometry for protein

identification. In parallel, we plan to systematically investigate the relationship between

protein glycation and aggregation by monitoring proteome-wide changes in protein

aggregation propensity induced by exposure to glyoxal. The combined study will allow

us to investigate whether AGEs are mechanistically linked to changes in protein

aggregation propensity. In the future, we plan to extend our work to primary cells and

tissue to study the targets of AGEs during aging and the effect of dietary interventions

and obesity on the extent and specificity of protein glycation.

Keyword: proteostasis, ageing, AGEs, glycation, mass spectrometry, target, aggregation

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(10) Analysis of Late-Onset Alzheimer’s Disease Using Patient-Specific In-Vitro Models

Ehrhardt T1, Jung M1, Bezold V2, Bork K2, Hartmann C1, Giegling I1, Rujescu D1 1 Martin Luther University Halle-Wittenberg, Department Psychiatry, Psychotherapy, and Psychosomatic Medicine

2 Martin Luther University Halle-Wittenberg, Department of physiological chemistry

Background and Aim of the Study: Alzheimer´s disease (AD) is the most common form of dementia.

Environmental and genetic factors contribute to the risk for AD, but the underlying disease mechanisms are poorly

understood. AD is a genetically complex disorder and shows heritability of up to 80%. Glia cells including astrocytes

and microglia are affected in AD patients contributing to the tau pathology and the accumulation of neurotoxic

amyloid as well. Genome-wide association studies (GWAS) allowed the identification of DNA variations associated

with an elevated risk for AD in recent years. Together with other groups, we identified a number of AD susceptibility

genes including CD33, SORL1, ABCA7, and TREM2, which highly recommends that the immune system plays a

major role in onset, progression and treatment of AD. Our research is part of the psychiatric university clinic in Halle

(UKH). Therefore, we have access to blood cultures of AD patients of our clinic suitable for the generation induced

pluripotent stem cells (iPS). Pluripotent stem cells can be differentiated into patient-specific microglia, which

represent the major part of the immune cells in the human brain. The diversity of microglia functions contributes to a

healthy immune system. Disturbed microglia function represents an important disease mechanism in AD. Activation

of inflammatory microglia leads to production of toxic substances and decreased phagocytosis. Besides the way of

activation, the polarization state mainly depends on the expression level of the two recently described surface marker

genes CD33 and TREM2. These genes are contrary and have to be in a homeostasis level for a controlled

phagocytosis. Mutations in this genes lead to an imbalance and can influence the amount of amyloid uptake.

Material and Methods: Blood was collected from AD patients carrying risk variants in AD susceptibility genes for the

generation of iPS cells. SNP variants in those genes were genotyped based on their potential function according to

literature and genomic localization (exonic, binding domain, or promoter affecting SNPs) and tested for association to

AD. Reprogrammed iPS cells carried the genetic background of a certain AD patient. Pluripotency was characterized

by alkaline phosphatase staining, the expression of pluripotency markers, and the differentiation into the three germ

layers. Crucial pluripotency markers are OCT4, SOX2, NANOG, KLF4, MYC, and LIN28. AD iPS cells were

differentiated into astrocytes and microglia and the differentiation status was characterized by the expression of

crucial glia cell markers. Further, AD susceptibility genes recently published in GWAS were analysed.

Results: The protein expression was successfully induced shown by IF and WB analysis. Cells were also screened

for the most efficient induction of the three germ layers and the induction of neural cell fates including glia cell fates.

The established AD-specific iPS cell lines from AD patients represent a powerful tool for the analysis of molecular

and cellular disease mechanisms. We established 4-step protocol for the generation of AD-specific microglia enabling

the focused analysis of AD-associated risk variants. The protocol was verified by morphology, FACS analysis, IF

analysis, and RNA expression of crucial microglia marker including hematopoetic lineage markers and the induction

of well-described microglia markers such as IBA1. The disease-specific analysis of the genetic background of AD

patients in iPS cells represents a completely new approach for the understanding of AD genetics and AD-associated

risk variants.

Discussion: Together, combining molecular genetics of AD for the investigation of risk variants and iPS cell

technology for the generation of patient- and disease-specific stem cells provide a promising approach to

characterize known disease mechanisms. These approaches contribute to the understanding of known disease

mechanisms and to the discovery of unknown disease aspects as well.

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(11) Proteasome deficiency in endothelial cells is causally linked to senescence

Odeta Meçe1, Govind M. Pai1, Nderim Kryeziu1, Claudia Ender1, Tilman Grune2, Regine Heller1

1Institute for Molecular Cell Biology, Centre for Molecular Biomedicine (CMB), Jena University Hospital,

2Deutsches Institut für Ernährungsforschung, Potsdam - Rehbrücke (DIfE)

Senescence is thought to contribute to endothelial dysfunction, which ultimately leads to

development of cardiovascular disease. The current project characterizes proteasomal

protein degradation in endothelial cells undergoing replicative or stress-induced

senescence in vitro or chronological ageing in vivo.

Our results demonstrate that human endothelial cells become senescent in vitro within

25 cumulative population doublings. Senescence was verified by increased number of

senescence-associated β-galactosidase (SA-β-gal)-positive cells, increased cell size and

granularity and upregulation of the p53/p21 pathway. In addition, protein carbonylation

and nitration as well as reduced GSH levels were observed. Studies on the expression

of proteasomal subunits revealed a decline of the β5 and β2 subunits in senescent

cells. In parallel, trypsin- and chymotrypsin-like activities were decreased. Similar

observations were made in primary endothelial cells derived from old mice (24 months

old) when compared to young mice (2 months old). Furthermore, treatment of early

passage human endothelial cells with H2O2 leading to premature senescence was

characterized by a decline of proteasomal trypsin- and chymotrypsin-like activities

without loss of β5 and β2 subunits.

Transient inhibition of proteasomal activity in endothelial cells by MG132 or bortezomib

led to premature senescence characterized by SA-β-gal-positive cells and increased

expression of p53 and p21. In parallel, persistent accumulation of carbonylated proteins

was observed indicating a role of oxidative stress in the development of senescence.

From these results we suggest a relationship between proteolytic insufficiency and

senescence, which may contribute to vascular ageing.

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(12) BENEFICIAL EFFECT OF ASCORBATE IN RENAL AGING

K Forman1,2, F Martínez1, M Cifuentes3,4, R Bertinat1, K Salazar1 and F Nualart1

1Center for Advanced Microscopy CMA BIO-BIO, Neurobiology and Stem Cell Laboratory. University of Concepcion,

Barrio Universitario s/n, Chile 2 Department of Nutrition and Dietetics, University of Concepcion, Barrio Universitario sn, Chile

3Department of Cell Biology, Genetics and Physiology, IBIMA, BIONAND, Andalusian Center for Nanomedicine and

Biotechnology, University of Malaga, Malaga, Spain. 4Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, (CIBER-BBN), Malaga, Spain.

Renal aging might be due to damage caused by inflammation, apoptosis and oxidative

stress. Vitamin C (VC) or ascorbic acid (AA) is a major antioxidant and plays an

essential role in defending against cellular damage. AA transport is mediated by high

affinity sodium-dependent transporters (SVCTs). The purpose of this study was to

evaluate the protective effect of AA in the kidney of male senescence-accelerated mice

(SAMP8). Also the influence of AA on the morphological parameters and on the renal

SVCT1 expression were investigated.

SAMP8 mice at 2 (young) and 9 (aged) months of age were used (n=15). One group

was treated with AA (10mg/Kg-IP-6 months). The expression of SVCT1 were analyzed

by conventional immunohistochemical/multi-labeling immunofluorescence analysis and

laser capture microdissection coupled to qRT-PCR. The expression of interleukin 1(IL-

1b), inducible nitric oxide synthase (iNOS) and survivin were measured by qRT-PCR.

The morphological study was performed using Hematoxylin-eosin staining.

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(13) Influence of glyoxal and methylglyoxal on mesenchymal stem cell differentiation

Susan Garbe1, Maria Schindler1, Juliane-Susanne Jung1, Alexander Navarrete Santos2, Tom Seeling1 and Anne Navarrete Santos1

1 Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Faculty of Medicine, Halle

(Saale), Germany 2Centre for Medical Basic Research, Martin Luther University Halle-Wittenberg, Faculty of Medicine, Halle (Saale),

Germany

Mesenchymal stem cells (MSC) differentiate into chondrocytes, bone, fat and muscle cells

and therefore play a crucial role in human development. About three quarters of the body

mass arises from MSC and after embryonic development they are important. The

dicarbonyls methylglyoxal (MGO) and glyoxal (GO) are inducers of advanced glycation end

products (AGEs). For AGEs an adverse influence in diabetes, renal dysfunction and vessel

stiffness has been described. The physiological concentrations of MGO and GO in human

plasma are 120 and 200 nM respectively. In the plasma of diabetic patients these

concentrations are up to six fold higher. Thus, we hypothesize that high MGO and GO

levels lead to failures in the cellular differentiation with consequences for mesenchymal cell

fate determination and multipotency.

The cell model C3H10T1/2, an immortalized multipotent mesenchymal stem cell line from

mouse embryos was used to investigate how MGO and GO affect the differentiation of

mesenchymal stem cells into adipocytes. Undifferentiated C3H10T1/2 cells were exposed

for 72 hours after reaching confluent stage to 50 and 100 µM of MGO and GO, alone and

simultaneously. The cellular formation of the AGEs MG-H1 (methylglyoxal-derived

hydroimidazolone 1) and carboxymethyllysine (CML) and the senescence and apoptosis

marker p21 was then analysed by flow cytometry. Treatment with 100 µM GO and MGO

significantly increased the expression of p21. CML and MG-H1 were detected after

exposure to MGO and GO alone and in combination. After then MGO/GO-treated

C3H10T1/2 cells were differentiated into adipocytes using induction media containing

insulin, dexamethasone and isobutylmethylxanthine (for 48 hours). The grade of adipogenic

differentiation was analysed by flow cytometry with Nile Red staining at day 13. An

exposure to MGO and GO for 72 hours did not significantly affect the differentiation of the

C3H10T1/2 cells into adipocytes.

We conclude that pathophysiological concentrations of MGO and GO affect mesenchymal

stem cells increasing AGE levels and p21 expression, whereas adipogenic differentiation

was not changed. Supported by DFG GRK 2155 ProMoAge

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(14) Neuroblastoma Metastasis Driven by Methylglyoxal

VS.Gnanapragassam1, M.Nagasundaram1, M.Scheer1 and R. Horstkorte1. 1 Institute of Physiological Chemistry, Martin Luther University Halle-Wittenberg, Germany.

Neuroblastoma is one of the most frequent neuronal malignancy, affects the young

children’s worldwide. Highly heterogeneous and most of these tumors progressed to

metastasis through multiple mechanisms [1]. Methylglyoxal is a side product of

glycolysis, their influence on neuroblastoma sialylation and metastasis, is not yet

investigated in detail [2,3,4].

Neuroblastoma cell lines were treated with methylglyoxal at micromolar concentrations.

Significant increase in advanced glycation end products (AGES) and its receptor RAGE

on treatment with higher concentration of methylglyoxal. Increased phosphorylated

nuclear factor kappa B (NFkB) and extracellular signal–regulated kinases (ERK) were

also observed. This led to strong influence of altered sialic acid expression. Further

more the proliferation and migration properties of these cells were also increased on

methylglyoxal treatment. This results illustrates, the altered role of methylglyoxal in

enhancing metastatic property through modulation of sialylation.

References

1. Aminzadeh S., et al. (2015) Energy metabolism in neuroblastoma and Wilms tumor. 2. Gnanapragassam VS., et al. (2014). Sialic Acid Metabolic Engineering: A Potential Strategy for the Neuroblastoma Therapy. PLOS ONE 11(4): e0154289. doi: 10.1371/journal.pone.0154289 . 3. Allaman I., et al. (2015). " Methylglyoxal, the dark side of glycolysis." Front Neurosci 9: 23. doi: 10.3389/fnins.2015.00023. 4.Sullivan LB., et al. (2016). Altered metabolite levels in cancer: implications for tumor biology and cancer therapy." Nat Rev Cancer. 16(11):680-693.

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(15) The anti-apoptotic properties of APEX1 in the endothelium

require the first twenty amino acids and converge on Thioredoxin-1

Christine Goy, Philipp Jakobs, Karin Aufenvenne, Yogi Altschmied, Jojo Haendeler

Heisenberg group - Environmentally induced Cardiovascular Degeneration, Clinical Chemistry, University of Duesseldorf,

and IUF-Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany

Introduction: The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a

disordered N-terminus, a redox and a DNA repair domain. APEX1 has anti-apoptotic

properties, which have been linked to both domains depending on cell type and experimental

conditions.

Objective: As protection against apoptosis is a hallmark of vessel integrity, we wanted to

elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and if, what

the underlying mechanisms are.

Material and Methods: Domain mapping of APEX1. Transient transfection of endothelial cells

with Superfect (Qiagen). Apoptosis measurements were performed with Annexin V,

Caspase3/7 ELISA and immunoblots for Caspase 3 cleavage. Lentiviral transduction of

endothelial cells with APEX(1-20) and Trx-1 were performed. Carotid ligation of mice and

immunostaining of aortas were performed.

Results: APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D cleavage,

potentially by binding to the unprocessed form. Diminished Cathepsin D activation results in

increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently

apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1,

because APEX1 (21-318) induces Cathepsin D activity, decreases Thioredoxin-1 protein

levels and thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-

20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1

via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In

an in vivo model of restenosis, which is characterized by oxidative stress, endothelial

activation and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the

endothelium of the carotids.

Conclusion: APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends

on the first twenty amino acids of APEX1. As proper function of the endothelium during life

span is a hallmark for individual health span, a detailed characterization of the functions of the

APEX1 N-terminus is required to understand all its cellular properties.

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(16) Establishment of a blood-brain barrier model from patient-derived

induced pluripotent stem cells for the analysis of Alzheimer’s disease

Carla Hartmann, Matthias Jung, Toni Ehrhardt, Ina Giegling, Dan Rujescu, Department

of Psychiatry, Psychotherapy, and Psychosomatic Medicine, Martin Luther University

Halle-Wittenberg, Halle/Saale, Germany

aCenter for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena & Jena University

Hospital, Hans-Knöll-Str. 2, D-07745 Jena, Germany bDepartment of Physiology, University of Pennsylvania, Philadelphia, USA

Dorsal root ganglia (DRG) neurons are important relay stations between the periphery

and the central nervous system and are essential for somatosensory signaling. Reactive

species are produced in a variety of physiological and pathophysiological conditions and

are known to alter electric signaling. Here we studied the influence of reactive species

on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp

method. Even mild stress induced by either low concentrations of chloramine-T (10 µM)

or low-intensity blue-light irradiation profoundly diminished action potential frequency

but prolonged single action potentials in wild-type neurons. The impact on evoked

action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-

resistant voltage-gated sodium channel NaV1.8 (NaV1.8–/–), the channel most important

for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T

caused a significant reduction of NaV1.8 peak current and progressively slowed down

inactivation at higher concentrations. Blue light had a smaller effect on amplitude but

slowed down NaV1.8 channel inactivation. The observed effects were less apparent for

TTX-sensitive NaV channels, rendering NaV1.8 as an important reactive-species-

sensitive component in the electrical signaling of DRG neurons, potentially giving rise to

loss-of-function and gain-of-function phenomena depending on the type of reactive

species and their effective concentration and time of exposure. The mechanisms

described here might be relevant for alterations in rapid electrical signaling observed in

aged tissue or under age-related pathophysiological conditions.

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(17) Delirium after cardiac surgery: non-invasive predictive parameters to identify patients at risk

Britt Hofmann1; Friederike Fee Barthel1; Pauline Eberlein1; Rüdiger Horstkorte2;

Andreas Wienke3; Hendrik Treede1; Andreas Simm

1Department of Cardiac Surgery, University Hospital Halle, Germany

2 Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Germany

3Institute of Medical Epidemiology, Biostatistics and Informatics, Martin-Luther-University Halle-Wittenberg, Germany

OBJECTIVES: Delirium after cardiac surgery is a problem with seriously consequences for

patients and healthcare. Preventive strategies from preoperative predictive non-invasive

assessments may reduce the incidence and severity of delirium. The present aim was to

explore preoperative non-invasive parameters for delirium in older patients undergoing cardiac

surgery with cardiopulmonary bypass.

METHODS: Patients (68.7 ± 9.3 years) scheduled for routine cardiac surgery were included (n

= 111). All patients were assessed pre-, peri- and postoperatively, and delirium was determined

using the confusion assessment method and the intensive care delirium screening checklist.

General cognitive and adaptive function was pre- and postoperatively evaluated with the clock

drawing test. The non-invasive parameters hand grip test (HGT) and skin autofluorescence

(sAF) were preoperatively recorded. All variables were analysed by uni-/multivariable logistic

regression.

RESULTS: Delirium was diagnosed in 24 patients (21.6%). Furthermore, univariable regression

analysis identified age (OR=1.09; 95%CI: 1.03-1.16; p=0.005), sAF (OR=3.9; 95%CI: 1.56-9.76;

p=0.004), non-dominant HGT (OR=0.93; 95%CI: 0.89-0.98; p=0.007) and dominant HGT

(OR=0.94; 95%CI: 0.89-0.98; p=0.005) as predictors of postoperative delirium. Finally, delirium

was independently associated with tissue glycation reflected by sAF (OR=2.82; 95%CI: 1.06-

7.49; p=0.038).

CONCLUSIONS: This study demonstrated for the first time a relationship between

postoperative delirium and the tissue glycation related skin autofluorescence. In addition,

postoperative delirium was associated with age and hand grip strength of the assessed cardiac

surgery patients.

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(18) Detection of free Advanced Glycation End Products in vivo

during Hemodialysis

C. Hohmann, Halle (Saale)/D, C.Henning, Halle (Saale)/D, K. Liehr, Halle (Saale)/D, Prof. Dr.

M. A. Glomb, Halle (Saale)/D

Prof. Dr. M. A. Glomb, MLU Halle-Wittenberg, Institute of chemistry – Food chemistry Kurt-Mothes Straße 2, 06120 Halle (Saale), D, [email protected]

Patients suffering from uremia (advanced chronic kidney failure) have to undergo hemodialysis on a

regular basis to eliminate catabolic products. Uremia is understood as a status of chronic inflammation

linked to oxidative and carbonyl stress on the patients’ organism1.

Numerous studies have documented accumulation of advanced glycation end products (AGEs) in the

bloodstream of such patients2. AGE free adducts can originate from proteolysis, direct modification of

free amino acids or digestion of Maillard modified proteins ingested with the daily diet.

The amount of AGE free adducts in blood plasma and dialysis fluid is therefore an applicable parameter

to characterize the inflammatory status of uremic patients.

A highly selective LC-MS/MS-method was used for the analysis of AGE levels both in plasma and dialysis

fluid. A broad range of 19 amino acid modifications could be identified and quantitated.

Due to their low molecular weight between 174,1 Da (N6-formyl lysine) and 428 Da (glucosepane) it is

expected that the free amino acid modifications are successfully eliminated by the dialysis procedure

using membranes with a typical 35-45 kDa molecular weight cut-off. Indeed, this assumption proved to be

valid for all but one of the target analytes, with a mean clearance rate of 75%. Only pyrraline as the single

exception showed an opposite behavior, increasing by 59% during the treatment.

To find an explanation for this phenomenon, in-depth studies were conducted. De novo formation in blood

after the intake of the corresponding precursor compound 3-deoxyglucosone with the dialysis fluid or

catalytic effects on formation by the dialysis membrane were ruled out. To determine the role of

exogenous sources like glycated food proteins, a controlled dietary study with 8 dialysis patients was

carried out. In the first of two sessions, the patients were offered bread rolls with a mean pyrraline content

of 25.2 μmol/serving. In the second session the patients were asked to eat solely before the treatment. As

a result, the food intake during dialysis was directly correlated to an increase in plasma and in dialysate

verifying the exceptionally high bioavailability of food born pyrraline3.

Keywords: dialysis treatment, blood plasma, dialysate, free AGEs

References (1) Miyata, T.; Wada, Y.; Cai, Z.; Iida, Y.; Horie, K.; Yasuda, Y.; Maeda, K.; Kurokawa, K.; van Ypersele de Strihou, C. Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int. 1997, 51, 1170–1181. (2) Agalou, S.; Ahmed, N.; Thornalley, P. J.; Dawnay, A. Advanced glycation end product free adducts are cleared by dialysis. Ann. N. Y. Acad. Sci. 2005, 1043, 734–739. (3) Hohmann, C.; Liehr, K.; Henning, C.; Fiedler, R.; Girndt, M.; Gebert, M.; Hulko, M.; Storr, M.; Glomb, M. A. Detection of Free

Advanced Glycation End Products in Vivo during Hemodialysis. J. Agric. Food Chem. 2017, acs.jafc.6b05013.

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(19) AGEs in hematopoietic development

Nicolas Huber, Karl Lenhard Rudolph

Advanced glycation end products (AGEs) are posttranslational protein modifications that

emerge from glycation, a non-enzymatic reaction between the protein and a sugar.

AGEs are reported to have negative effects on proteins, cells, tissues and organisms

and are linked to several age-related diseases such as Glycation is a non-enzymatic

reaction between a reducing sugar and a protein, which leads to the formation of

advanced glycation end products (AGEs). These posttranslational modifications are

often seen as protein damage as they can lead to aggregation, degradation or loss of

function. AGEs are linked to several age-related pathologies such as Alzheimer’s

disease, diabetes, inflammation signaling and cardiovascular diseases among others.

We investigate AGEs in the murine hematopoietic system, a tissue strongly relying on

stem cell function and differentiation which is compromised in older individuals. We find

that throughout hematopoietic development, populations show different AGE levels,

depending on their lineage and differentiation stage. We find these population-

dependent levels to be unchanged in old age and in dietary restriction, which indicates

that these levels are conserved and potentially inevitable or necessary. The differences

in AGE levels point towards a possible relationship between AGEs and differentiation

within the hematopoietic system. The question to be answered is if differentiation drives

AGE levels or if AGEs could actually contribute to differentiation. This would argue for

an important role of AGEs and could contribute to rethink the dogma that renders AGEs

only harmful to the organism.

Though focused on aging, the field of AGE research neither concentrates on stem cells

nor the hematopoietic system, thereby neglecting their importance. Our studies serve to

gain a better understanding of the biological role of glycation during aging and in stem

cells, starting with the hematopoietic system.

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(20) Non-enzymatic modifications of the mineralocorticoid receptor during vascular aging

Ralf Hübschmann, Stefanie Ruhs, Claudia Großmann

Julius-Bernstein-Institut für Physiologie Halle

Numerous clinical studies showed that the mineralocorticoid receptor (MR), a ligand-

dependent transcription factor, mediates pathophysiological processes like

inflammation, fibrosis and endothelial dysfunctions in the cardiovascular system. An

important aspect of the deleterious MR effects is that the receptor and its natural ligand

aldosterone per se are not capable of eliciting pathophysiological effects but requires a

certain micro-milieu. Several studies indicate that a parainflammatory micro-milieu

created by a shift in redox state and/or altered level of oxidative and nitrosative stress

(ROS/RNS) represents a possible trigger to induce pathophysiological MR effects.

During ageing, vascular cells are increasingly exposed to such a parainflammatory

micro-milieu which then augments pathophysiological MR signaling, leading to age-

associated inflammatory processes (vasculopathy). Our experiments show that

nitrosative stress influences genomic MR activity. Whereas nitric oxide (NO) attenuates

aldosterone-induced transcriptional MR activity by reducing its binding to DNA

elements, peroxynitrite (ONOO–) acts as a ligand-independent MR activator. When

exploring the underlying molecular mechanism, we found that activation of the MR leads

to posttranslational modification of the receptor and that PTM of the MR may also occur

during conditions involving enhanced nitrosative stress. Co-immunoprecipitation and

Western blot experiments show an increased S-nitrosylation level of the MR after NO

treatment whereas ONOO– stimulation leads to an elevated amount of nitrated MR

tyrosines. In silico predicted modified cysteine and tyrosine residues will be validated by

mass spectrometry and then characterized by performing site-directed mutagenesis

experiments followed by functional analyses of mutated MR compared to wild type

receptor. Overall, these experiments will help to understand pathophysiological MR

signaling during nitrosative stress conditions which occur during ageing.

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(21) Stem cells modelling Alzheimer's disease for the analysis of molecular aging of the brain

Matthias Jung, Toni Ehrhardt, Carla Hartmann, Jovita Schiller, Ina Giegling, and Dan Rujescu

Department of Psychiatry, Psychotherapy, and Psychosomatic Medicine, Martin Luther University Halle-Wittenberg

Aim: Alzheimer’s disease (AD) is the most common form of dementia caused by environmental and

genetic factors. The majority of patients are suffering from late onset AD in persons older than

65 years. One characteristic of AD is the accumulation of amyloid-β (Aβ) in the brain. Recently,

19 loci in the DNA of AD patients were associated with AD including CLU. CLU has been

described as a potential regulator of brain function during aging. To investigate molecular aging

mechanisms in AD, we established stem cell-derived cerebral in vitro models for the patient-

specific analysis of psychiatric genetics.

Methods: Episomal vectors were used for the generation of induced pluripotent stem (iPS) cell lines from

patient-specific B-lymphoblastoid cell lines (B-LCLs). Pluripotency was verified and a screening

protocol ensured a high neuronal differentiation potential towards neural and glial lineages.

Cells were also applied to generate self-renewing neural stem cells (NSCs) suitable to generate

neurons and astrocytes in 2D and 3D models.

Results Permanent iPS cell lines and NSCs were characterized by morphology and the presence of

stem cell-specific marker genes including OCT4 and NANOG in iPS cells and SOX2 and PAX6

in NSCs. Stemness markers were analyzed whether they represent appropriate aging markers.

NSCs were successfully differentiated into neurons and astrocytes. Astrocyte-specific markers

including EAAT1 demonstrated efficient astrocyte differentiation. The efficient differentiation of

mature neurons was shown by the presence of glutamate and GABA receptors. Mitochondrial

respiration was elevated in differentiated neurons. Patch clamp analysis verified the functionality

of GABAergic and glutamatergic neurons and astrocytes. 3D organoids obtained from NSCs

showed the expression of cortical markers such as TBR1 mimicking the developing human

cortex. Cells obtained from young and old patients showed different properties.

Together, stem cells modelling AD represent a powerful tool to study molecular aging of the

brain.

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(22) Are AGE-modified amino acids proteinogenic?

Astrid Junk1,2, Anne Kummer1, Patrick Winterhalter1,2, Tim Baldensberger2,3, Marcus

Glomb2,3, Andreas Simm1,2

1Clinic of Heart Surgery, Martin-Luther-University Halle-Wittenberg, Halle (Saale),

2ProMoAge, GRK 2155,

3Institute of food chemistry, Martin-Luther-University Halle-Wittenberg, Halle (Saale)

The characteristic western diet contains heated and highly processed proteins. These

processes lead to various artificial modifications e.g the formation of Advanced

Glycation Endproducts (AGEs). The reaction is defined as a non-enzymatic addition of

reactive carbohydrates e.g. sugars to amino acids. The endogenous formation of AGEs

is a part of the normal ageing metabolism. High levels of AGEs in tissues and body

fluids can become pathogenic due to their ability to promote oxidative stress and

inflammation. Current research could show, that only a part of the absorbed AGEs are

excreted via the kidneys after 48 hours, the fate of the non-excreted AGEs remains

undetermined.

Within this project, the reuse of single AGE-modified amino acids, e.g.

Carboxymethyllysine (CML), in protein translation is tested.

In an in vitro translation approach based on Hela lysates and a GFP-expression vector,

the addition of CML induce an increase influorescence without any change in protein

amount of the expressed target protein. Control experiments exclude effects of pH,

charge and molarity as a cause of modified fluorescence. Furthermore, due to a total

amino acid analysis of the produced proteins, incorporation of CML could be detected.

Thus, this first results indicates hint at a low rate of translational incorporation of CML.

The next step will be a mass spectrometric analysis to clarify which amino acids are

substituted. In addition, cells will be fed with CML-containing-dipeptides to show a

possible impact on the in-vivo translation.

Our data indicate on a possible role of nutrition related AGEs on in-vivo aging by

incorporation into newly synthesized proteins within translation.

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(23) Two putative selenium binding proteins as modulators of C.

elegans stress response and life span

Karl Köhnlein1, Nadine Urban1, Holger Steinbrenner1, Christoph Kaether2, Lars-Oliver

Klotz1

1Institute of Nutrition, Department of Nutrigenomics, Friedrich-Schiller-Universität Jena, Germany

2Leibniz Institut für Alternsforschung-Fritz Lipmann Institut, Jena, Germany

Selenium-binding proteins do not contain selenium in the form of selenocysteine or

selenomethionine but as inorganic selenium bound to the protein. The C. elegans

genome encodes only one selenocysteine-containing selenoprotein, TrxR-1. However,

at least two ORFs encode putative selenium-binding proteins, CeSELENBP1 and

CeSELENBP2. These are 54% and 36% homologous to human selenium-binding

protein-1 (SELENBP1), respectively, and both contain a cysteine residue hypothesized

to bind selenite as in SELENBP1.

Considering the role of selenium in antioxidant defense, we hypothesized that

CeSELENBP1 and CeSELENBP2 may modulate the response of C. elegans to

oxidative stress. Unexpectedly, life-long knock-down of either of the genes significantly

increased life span and stress resistance to the redox cycler paraquat.

DAF-16 and SKN-1 are key players in the C. elegans stress response that are known to

mediate many of the published life-extending effects of chemical compounds,

phytochemicals and nutrition regimens. However, RNAi against CeSELENBP1 and

CeSELENBP2 significantly increased life span of the above mutants to an extent similar

to that found in wild type worms exposed to RNAi targeting either of the putative

selenium binding proteins. This suggests that neither DAF-16 nor SKN-1 are involved in

life span extension elicited by knock-down of CeSELENBP1 and CeSELENBP2.

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(24) A genetically encoded indicator of methionine oxidation

Kuldyushev N.A., Schönherr R., Heinemann S.H.

Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, Hans-Knöll-Str. 2, 07745 Jena, Germany [email protected], [email protected]

Post-translation modifications are essential in the fine-tuning of protein signaling

networks. During aging, the levels of some post-translational modifications are

changing, thereby affecting the functions of modified proteins. Methionine oxidation

to methionine sulfoxide (Met(O)) is a post-translational modification that can appear

unspecifically during an exposure of methionine to oxidative stress, such as

occurring in aged organisms and in a number of degenerative diseases and

pathologies. The importance of methionine oxidation is further emphasized by the

fact that virtually all organisms harbor a set of enzymes – methionine sulfoxide

reductases (MSRs) – that specifically reduce free and protein-based methionine

sulfoxide back to methionine.

Here we introduce a genetically encoded fluorescent sensor for methionine

oxidation. Variants based on green fluorescent protein (GFP) were designed to

provide a ratiometric fluorescence signal reporting on the degree of methionine

oxidation. Such fluorescent proteins were produced in a bacterial expression

system and their properties were studied under in vitro conditions in a cuvette.

Experimental oxidation with H2O2, chloramine T, or hypochloric acid resulted in

robust changes in the fluorescent ratio F400/F470. Incubation with recombinantly

produced MSRs indicated that the sensor is not a substrate for the enzymes. Upon

expression in mammalian cells (HEK293T), the sensor produced ratiometric

fluorescence signals suited for single-cell photometry and life-cell imaging to

monitor intracellular methionine oxidation events. This study will contribute to a

better understanding of how oxidative modification levels change during aging and

disease states.

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(25) Potential of glycated beta-Amyloid as an early diagnostic marker

for Alzheimer's disease

Anne Kummer1, Alexander Navarrete Santos2, Hendrik Treede1, Andreas Simm1,2

1 Clinic for Heart Surgery, Martin-Luther-University Halle-Wittenberg, Halle (Saale)

2 Center for Medical Basic Research, Martin-Luther University Halle-Wittenberg, Halle (Saale)

Alzheimer’s disease (AD) is an age-related, neurodegenerative disorder that poses a

growing challenge especially due to the ongoing demographic transition. Furthermore,

AD is not curable and medication is only capable of delaying or slowing the disease

progression. Hence, an early diagnosis is of utmost importance.

One patho-mechanism of Diabetes, a major risk factor for AD, is the carbohydrate-

induced glycation of proteins, forming long-lasting and non-reversible advanced

glycation end products (AGEs). Beta-Amyloid (Abeta), a polypeptide implicated in AD,

exhibits increased cytotoxicity in its glycated form. Thus, this project aims to clarify

whether glycated, soluble Abeta is a suitable early marker for the diagnosis of AD.

For this purpose, an existing Abeta-based, flow cytometric assay was modified to detect

glycated forms of the soluble polypeptide in cerebrospinal fluid (CSF). In parallel, a

recombinant standard was established to also facilitate the quantification of the modified

Abeta via flow cytometry. In principle, the carboxymethyl-lysine containing, recombinant

standard of Abeta can be detected by this assay. Currently, the reproducibility is

assessed for this assay and alternative detection methods of glycated Abeta are

considered.

Furthermore, immunoprecipitation of Abeta and subsequent mass spectrometric

analyses will be utilized to identify the glycated Abeta-forms predominantly present in

CSF of AD-patients in comparison to healthy controls.

The ultimate goal, however, is to establish a diagnostic assay for the early diagnosis of

Alzheimer’s based on the obtained results.

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(26) Assessment of murine gut motility under stress conditions

Johanna Langner

Gut failure and the development of a megacolon emerging from impaired gut motility are

significant pathological factors in severe sepsis. Furthermore, imbalance of the gut

microbiome, such as excessive C. difficile growth following antibiotics treatment, can

cause life-threatening complications. In this project molecular mechanisms underlying

gut dysmotility, which can lead to gut failure and even death, are to be investigated.

Gut motility strongly relies on rhythmic depolarization of the smooth muscle cells

(SMCs), which at least in part are triggered by specialized SMCs with pacemaking

function: Interstitial Cells of Cajal (ICC-My). Besides measuring the mechanical force

exerted by the smooth muscle layer, the membrane potential (Vm), the intracellular Ca2+

concentration ([Ca2+]i), or the intracellular redox potential are potential parameters for

the characterization of gut motile activity. To measure fluctuations of these parameters

in SMCs and ICC-My, genetically encoded fluorescence sensors reporting on Vm,

[Ca2+]i, or the formation of disulfide bonds are developed and examined.

Electrical rhythm generation requires the concerted activity of ion channels, which may

be relevant targets for stress factors affecting gut motility. Here we concentrate on K+

and Cl– conductances that are important regulators of the electrical activity in ICC-My.

In particular we examine how the Ca2+-activated Cl– channel TMEM16A operates in

ICC-My under physiological conditions and after exposure to oxidative stress (e.g.

reactive species, heme, Fe2+) and low molecular weight compounds which are released

from the microbiome under control and disease conditions.

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(27) The Gid/CTLH Ubiquitin Ligase Complex: A Novel Cilia-

Associated Regulator of Autophagy

Huaize Liu1,6, Jie Ding1, Pablo Villavicencio-Lorini2, Peter Walentek4, Gerd Hause5,

Thomas Hollemann1, Thorsten Pfirrmann1,6,7

1 Martin-Luther University Halle-Wittenberg, Institute of Physiological Chemistry, Halle, Germany

2 Martin-Luther University Halle-Wittenberg, Institute of Human Genetics, Halle, Germany

4 University of California at Berkeley, Division of Genetics, Genomics and Development, Centre for Integrative

Genomics, Berkeley, USA 6 These authors contributed equally to the work

7 Address correspondence to: [email protected]

Keywords: CTLH complex / ubiquitin/ ciliogenesis/ ciliopathies/ Xenopus laevis/ autophagy/ ageing

Pharmacological or genetic manipulations that increase life span in model organisms

often stimulate autophagy by the specific inhibition of the mTOR pathway. Cilia are

highly evolutionary conserved organelles involved in embryonic development, sensing

of extracellular milieu, and a variety of signal transduction pathways. Recent studies

propose a dual-interplay between cilia function and autophagy that regulates metabolic

homeostasis, e.g. elongated primary cilia have a reduced mTOR activity and an

increased autophagic flux. Here, we describe the function of an evolutionary conserved,

cilia associated ubiquitin ligase complex as a novel modulator of mTOR signaling and

autophagy. In Saccharomyces cerevisiae, the Gid/CTLH complex regulates the

metabolic switch from gluconeogenesis to glycolysis by targeting key enzymes of

gluconeogenesis for proteasomal degradation. However, its function and substrates in

vertebrates remains enigmatic. We provide evidence that the Gid/CTLH complex has a

function during the formation of both motile and primary cilia. The subunit Rmnd5a co-

localizes with the basal body of primary cilia in NIH3T3 cells, and Rmnd5a loss of

function leads to the elongation of primary cilia. Interestingly, this correlates with an

increase in autophagic flux and attenuated mTOR activity. Together, our data suggests

an intimate function of cilia-associated regulation of autophagy and metabolic

homeostasis that most likely is mediated by an unknown substrate of the Gid/CTLH

complex. We hypothesize that the primary cilium is a nutrient sensor that regulates

metabolic homeostasis and nutrient signaling via the Gid/CTLH complex. Thus the

Gid/CTLH complex emerges as a promising new target to pharmacological alter mTOR

activity and its known effects on longevity.

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(28) Age related glycation hotspots in plant proteome

Tatiana Bilova,1,2 Tatiana Mamontova,1,3 Gagan Paudel,1,4 Dominic Brauch,4 Svetlana Milrud,2,3

Rico Schmidt,5 Elena Tarakhovskaya,2 Nikita Shilyaev,3 Galina Smolikova,2 Alain Tissier,6

Thomas Vogt,6 Claudia Birkemeyer,4 Andrea Sinz,5 Wolfgang Brandt,1 Ludger A. Wessjohann,1

and Andrej Frolov,1

1Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry (Halle/Saale),

2Departments of Plant

Physiology and Biochemistry and 3

Biochemistry, St. Petersburg State University (Russia), 4Faculty of Chemistry and

Mineralogy, Universität Leipzig,

5Institute of Pharmacy, Martin-Luther Universität Halle-Wittenberg and

6Department

of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry (Halle/Saale).

Advanced glycation is referred to as a non-enzymatic reaction of proteins with α-dicarbonyl

intermediates of sugar autoxidation, glycoxidation and lipid peroxidation. It is a factor of protein

damage resulting in accumulation of advanced glycation end products (AGEs) in mammalian

tissues. In this context, AGEs are well-known markers of ageing. In comparison to mammals,

plants are characterized with higher tissue contents of reactive carbohydrates and increased

rates of oxidative processes. This might result in enhanced carbonyl stress, and, hence, higher,

in comparison to mammals, levels of protein glycation. Indeed, recently, rich constitutive plant

glycation patterns were characterized by our group [1]. However the susceptibility of plant

proteins to age-related glycation was not addressed so far. Therefore, here we consider this

problem in the context of possible mechanisms underlying age-related glycation using 7-12-

week-old A. thaliana plants and methods of biochemistry, molecular biology, metabolomics and

proteomics. Although qualitative glycation patterns were relatively stable in time, multiple

proteins demonstrated strong increase of glycation (up to 28 fold) by specific sites, i.e. glycation

hotspots were observed [2]. Many up-glycated proteins were involved in protein metabolism and

transcriptional regulation. Four of them contained AGE-modifications in their functional domains

that could affect protein functionality. The identified glycation hotspots dominated with arginine-

derived hydroimidazolones, originating from glyoxal (Glarg) and methylglyoxal (MG-H). Analysis

of glycation-specific sequence motifs revealed ionic (K, R, E, D) and non-ionic (L,V, A, P)

residues in close proximity to the glycation hotspots. Homology modeling of glycated proteins

revealed glutamyl and aspartyl residues in close proximity to the hotspots (at the distance less

than 5Å), that might indicate their enhancement effect on AGE formation.

References

1. Bilova T, Lukasheva E, Brauch D, Greifenhagen U, Paudel G, Tarakhovskaya E, Frolova N, Mittasch J, Balcke GU, Tissier A, Osmolovskaya N, Vogt T, Wessjohann LA, Birkemeyer C, Milkowski C, Frolov A (2016). A Snapshot of the Plant Glycated Proteome: Structural, Functional and Mechanistic Aspects. J. Biol. Chem., 291(14):7621-36.

2. Bilova T., Paudel G., Shilyaev N., Schmidt R., Brauch D., Tarakhovskaya E., Milrud S., Smolikova G., Tissier A.,

Vogt T., Sinz A., Brandt W., Birkemeyer C., Wessjohann L. A., Frolov A. (2017). Global Proteomic Analysis of

Advanced Glycation End Products in the Arabidopsis Proteome Provides Evidence for Age-related Glycation Hotspots. J. Biol. Chem., 2017 (First Published on June 13, 2017, accepted jbc.M117.794537. doi:10.1074/jbc.M117.794537)

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(29) Advanced Glycation End Products, oxidative stress and Glyoxalase expression in breast cancer

Norbert Naß*1, Ludwig Andreas1, Atanas Ignatov2, Johannes Haybäck1 and Thomas

Kalinski1

1: Otto von Guericke University Magdeburg, Department of Pathology, Leipziger Str. 44, D-39120 Magdeburg, Germany; 2: Otto von Guericke University Magdeburg, Department of Obstetrics and Gynecology, Gerhart-Hauptmann Str. 35, 39108 Magdeburg, Germany * Contact: [email protected]

Proteins modified by Advanced Glycation End products (AGEs) are well documented

biomarkers for ageing and several degenerative diseases. AGE-modification has

significant effects on the stability of the extracellular matrix thus contributing to age-

related stiffening of organs. Signaling molecules and enzymes can be inactivated by

glycation and AGEs are recognized by the receptor for AGEs (RAGE), which leads to

an activation of the NF-κB transcription factor. Due to metabolic changes, especially the

Warburg effect and oxidative stress, tumors often accumulate significant amounts of

AGE-modified proteins. Thus, cancer cells need to detoxify glycating aldehydes by

means of glyoxalases, namely glyoxalase-1 (GLO1) and DJ-1 (PARK7).

We here investigated the contribution of the AGE-system to breast cancer biology and

prognosis. The accumulation of the AGE carboxymethyl-lysine (CML) was

retrospectively investigated by immunohistochemistry in a cohort of about 200 breast

cancer patients. High CML-accumulation correlated with estrogen receptor expression

and age and was related to unfavorable prognosis in estrogen receptor negative

tumors. In cell culture experiments, GLO1 turned out to be important to confer α-oxo-

aldehyde resistance. Also, the toxicity of α-oxo-aldehydes correlated with adaption of

estrogen receptor positive cell lines to the selective estrogen receptor mediator

tamoxifen and such cells showed an altered regulation of NF-kB signaling.

Altogether these data suggest an important role of the AGE-system for breast cancer

biology. Especially glyoxalases might represent effective targets for the treatment of this

disease.

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(30) Dicarbonyls induce senescence of human vascular endothelial

cells

Alexander Navarrete Santos§, Kathleen Jacobs#, Andreas Simm#§, Nicole Glaubitz#;

Rüdiger Horstkorte*, Britt Hofmann#

§Centre for Medical Basic Research of the Martin-Luther-University Halle-Wittenberg, Germany

#Department of Cardiac Surgery, University Hospital Halle, Germany

*Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Germany

Rationale: Glyoxal (GO) and Methylglyoxal (MGO) are two dicarbonyls involved in the

formation of advanced glycation end products (AGEs). Endothelial cells in the vessels

are in constant contact with circulating AGEs and dicarbonyls. With this project, we

aimed to elucidate the effect of GO and MGO on primary human vascular endothelial

cells (HVECs).

Methods: Graft material from patients with coronary heart disease was used as HVECs

source. HVECs were treated with different concentrations of GO and MGO. -

Galactosidase related senescence activity and cell morphology were analyzed. AGEs

as well as p21 protein expression, glyoxalase-I expression and oxidative stress were

detected.

Results: We here provide evidences that GO and MGO induce senescence in primary

HVECs. Mechanistically GO and MGO induce senescence by increasing the ROS

production, the expression of p21, the accumulation of AGEs and the arrest of HVECs

in the G2 cell cycle phase. Aminoguanidine- a dicarbonyl scavenger- abrogated the

effect of GO and MGO.

Conclusion: Our data are relevant as they suggest that in diseases with elevated

dicarbonyl concentrations, deleterious effects on the endothelium and the development

of vascular dysfunction must be expected. On the other hand, treatment of patients with

dicarbonyl scavenger could prevent this.

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(31) Age and diet affect the intestinal crypt proteome

Nadja Gebert1, Joanna Kirkpatrick1, Jerome Korzelius1, Henri Jasper1,2 and

Alessandro Ori1

1 Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Jena, Germany

2 Buck Institute for Research on Aging, Novato, USA

The small intestine is responsible for nutrient sensing and absorption, and it is one of

the most important interfaces between the environment and our body. During

aging, deregulation of intestinal stem cell (ISC) activity leads to loss of epithelial barrier

function, food malabsorption and dysbiosis of commensal bacteria.

In order to investigate both cell intrinsic and extrinsic factors influencing ISC aging, we

isolated crypts and stem cells from the small intestine of mice and D.

melanogaster midgut. We compared proteomic profiles of tissues and cells from

different age groups using state-of-the-art mass spectrometry. We found proteome

signatures in the intestinal epithelium, which indicate that aging affects metabolic

networks, stem cell proliferation, and epithelial immune responses. Of note, some of

these aging-associated alterations (e.g., immune modulatory responses) are reverted

by dietary restriction (DR), a health span extending intervention conserved across

species. In particular, we found that both aging and DR influence the expression of a

key enzyme in ketone body metabolism, and demonstrate that perturbation of its activity

as well as the exogenous supplementation of ketone bodies can influence ISC

regeneration in organoid cultures. Our data demonstrate how aging and dietary

intervention can modulate metabolic networks and influence stem cell activity by altering

the concentration of metabolites in the niche.

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(32) Integrative analysis of age related changes in the transcriptome of

Caenorhabditis elegans

Tsimafei Padvitski, Adam Antebi, Andreas Beyer

To obtain insight into age-related changes of multicellular organism, we studied global

transcriptome of C. elegans under normal aging using RNAseq time-series experiment.

The data was obtained in RNAseq experiment on synchronized culture of wild-type

C.elegans sampled at day 0, 7, 14 and day 21 (all stages but the first are

postreproductive). Then we performed a gene-level differential expression analysis and

a subsequent functional annotation. The results support the theory that ageing is a

highly stochastic process. At the same time, they also show that specific processes and

pathways are more vulnerable to the stochastic changes.

Next, we used time-series clustering to obtain gene sets that represent transcriptome

modules with distinct temporal dynamics in aging. Finally, we questioned if these

transcriptome modules can be controlled by specific transcription factors. To answer

this question, we integrated public Chip-seq data from modENCODE with the results of

the time-series clustering using a simple statistical framework. We identified a

consistent set of TFs, all have been previously described as implicated in aging.

Interestingly, the same set of TFs can presumably control behavior of genes with

different dynamics during aging.

To summarize, we characterized in details age-related changes in the global

transcriptome of C. elegans and showed that integration of gene expression with Chip-

seq data allows the prediction of TFs that are capable of modulating the lifespan of C.

elegans.

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(33) High risk of malnutrition is associated with low muscle mass in

older hospitalized patients - a prospective cohort study

Vincent D. Pierik BSc1, Dr. Carel G.M. Meskers2, Jeanine M. Van Ancum MSc1,3, Siger

T. Numans BSc1, Sjors Verlaan MSc1,4, Kira Scheerman MSc5, Dr. Roeliene C.

Kruizinga1, Prof. Dr. Andrea Maier3,6 *

1 Department of Internal Medicine, Section of Gerontology and Geriatrics, VU University Medical Center, Amsterdam, The

Netherlands

2 Department of Rehabilitation Medicine, VU University Medical Center, Amsterdam, The Netherlands

3 Department of Human Movement Sciences, MOVE Research Institute Amsterdam, VU University, Amsterdam, The

Netherlands

4 Nutricia Research, Nutricia Advanced Medical Nutrition, Utrecht, The Netherlands

5 Division I, VU University Medical Center, Amsterdam, The Netherlands

6 Department of Medicine and Aged Care, Royal Melbourne Hospital, University of Melbourne, Melbourne, Australia

Background: Malnutrition, low muscle strength and muscle mass are highly prevalent in older

hospitalized patients and associated with adverse outcomes. Malnutrition may be a risk factor for

developing low muscle mass. We aimed to investigate the association between the risk of

malnutrition and 1) muscle strength and muscle mass at admission and 2) the change of muscle

strength and muscle mass during hospitalization in older patients.

Methods: The EMPOWER study included 378 patients aged seventy years or older who were

acutely or electively admitted to four different wards of an academic teaching hospital in

Amsterdam. Patients were grouped into low risk of malnutrition and high risk of malnutrition

based on the Short Nutritional Assessment Questionnaire (SNAQ) score and were assessed for

hand grip strength and muscle mass using hand held dynamometry respectively bioelectrical

impedance analysis (BIA) within 48 hours after admission and at day seven, or earlier at the day

of discharge. Muscle mass was expressed as skeletal muscle mass, appendicular lean mass, fat

free mass and the skeletal muscle index.

Results: The mean age of the patients was 79.7 years (SD 6.39), 48.9 % were female. At

admission, being at high risk of malnutrition was significantly associated with lower muscle

mass (Odds Ratio, 95 % CI, 0.90, 0.85-0.96), but not with muscle strength. Muscle strength and

muscle mass did not change significantly during hospitalization in both groups.

Cconclusion: In older hospitalized patients, a high risk of malnutrition is associated with lower

muscle mass at admission, but not with muscle strength nor with change of either muscle

strength or muscle mass during hospitalization.

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(34) Attempts to remove AGEs from human plasma using polysterene-

divinylbenzene polymer adsorbent

Veronika Piskovatska, Alexander Navarrete Santos, Andreas Simm

Martin-Luther-University Halle-Wittenberg, University Clinic for Cardiac Surgery

Background: Advanced glycation end products (AGEs), forming in vivo under

conditions when dicarbonyl compounds react with proteins, are shown to have a

significant role in ageing-associated diseases. Pharmacological agents, reducing AGEs

levels in plasma by inhibiting certain stages of glycation, demonstrated clinical benefits

in elderly patients with different conditions (metformin, aspirin, ACEi).

The aim of the project was to evaluate the binding capacity of CytoSorb adsorber,

currently approved for reduction of extremely elevated cytokine levels in intensive care

units patients. Adsorber efficiently binds proteins within 5-60 kDa range in

concentration-dependent manner.

Materials and methods: AGE-modified human plasma was put through centrifuge

columns, packed with CytoSorb material in buffer. Efficiency of AGEs binding was

compared in CytoSorb column in parallel with immobilized boronic acid resin column,

which is described to specifically bind AGEs. Glycated proteins from plasma samples

before and after treatments, washouts and elutions were analyzed by immunoblot.

Autofluorescence of AGEs at different wavelengths was evaluated via fluorescent plate-

reader.

Results: CytoSorb columns efficiently binds CML, pentosidine, MGH1, CEL and AGE-1

(glucose-modified) glycated proteins, qualitative immunoblot results demonstrated that

polymer-based column removes AGEs from plasma in comparable extent as the

boronic acid procedure. Column treatment decreased the autofluorescence of plasma

samples at 360/440 nm, corresponding to reduction of fluorescent AGEs. Repeated

plasma purification with polymer-filled column also increased the extent of AGEs

removal from the modified plasma samples.

Conclusion: the extent of binding, as well as specificity towards different kinds of AGEs

are yet to be identified, as well as specificity of the selected adsorbent system towards

variable protein posttranslational modifications of clinical relevance.

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(35) Individual variance in resting metabolic rate and energy balance

in geriatric outpatients with mobility problems

Esmee M. Reijnierse1, Sjors Verlaan2, Marijke C. Trappenburg2,3, Carel G.M.

Meskers4,5, Marian A.E. de van der Schueren6,7, Andrea B. Maier1,5

Affiliations 1Department of Medicine and Aged Care, The Royal Melbourne Hospital, The University of Melbourne, Australia

2Department of Internal Medicine, Section of Gerontology and Geriatrics, VU University Medical Center, Amsterdam,

The Netherlands 3Department of Internal Medicine, Amstelland Hospital, Amstelveen, The Netherlands

4Department of Rehabilitation Medicine, VU University Medical Center, Amsterdam, The Netherlands

5Department of Human Movement Sciences, MOVE Research Institute Amsterdam, Vrije Universiteit, Amsterdam,

The Netherlands 6Department of Internal Medicine, Section of Nutrition and Dietetics, VU University Medical Center, Amsterdam, The

Netherlands 7Department of Nutrition and Health, Faculty of Health and Social Studies, HAN University of Applied Sciences

Nijmegen, The Netherlands

Rationale: In clinical practice, optimization of physical and nutritional intervention (“tailored

therapy”) requires patient specific data on energy expenditure and nutritional intake tailored to

the individual requirements; these data are generally lacking. This study aimed to explore the

nutritional needs of geriatric outpatients regarding energy expenditure and energy balance.

Methods: This inception cohort of older adults referred to the geriatric outpatient clinic of the

Center of Geriatric Medicine Amsterdam, VU University Medical Center included 26 outpatients.

Geriatric outpatients underwent standardized phenotyping based on regular care and additional

nutritional (indirect calorimetry to measure resting metabolic rate (RMR), three day food diary)

and physical activity assessments (accelerometry). Next to indirect calorimetry, RMR was

estimated using the equation by the World Health Organization based on age, sex, body weight

and height. Total energy expenditure (TEE) was calculated by RMR times the physical activity

level. Energy balance was defined as TEE versus the energy intake.

Results: A high individual variance was found in both RMR and energy balance. Mean

difference between the actual measured RMR and the estimated RMR was 234 kcal/day on a

population level. On the individual level, estimated RMR underestimated the actual measured

RMR by more than 10% in more than half of the outpatients. Mean difference between TEE and

energy intake was 429 kcal/day. Half of the outpatients had an energy consumption lower than

their needs by more than 10%.

Conclusions: RMR should be measured objectively due to the high underestimation of

estimating RMR. In geriatric outpatients, it is important to individually quantify the nutritional

needs.

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(36) Lack of knowledge and availabiliy of diagnostic equipment hinder

the diagnosis of sarcopenia and its management

Esmee M. Reijnierse1, Marian A.E. de van der Schueren2,3, Marijke C. Trappenburg4,5,

Marjan Doves6, Carel G.M. Meskers7,8, Andrea B. Maier1,8

Affiliations 1Department of Medicine and Aged Care, University of Melbourne, Melbourne, Australia

2Department of Internal Medicine, Section of Nutrition and Dietetics, VU University Medical Center, Amsterdam, The

Netherlands 3Department of Nutrition and Health, Faculty of Health and Social Studies, HAN University of Applied Sciences,

Nijmegen, The Netherlands 4Department of Internal Medicine, Section of Gerontology and Geriatrics, VU University Medical Center, Amsterdam,

The Netherlands 5Department of Internal Medicine, Amstelland Hospital, Amstelveen, The Netherlands

6Institute of Human Movement Studies, Faculty of Health Care, University of Applied Sciences Utrecht, Utrecht, The

Netherlands 7Department of Rehabilitation Medicine, VU University Medical Center, The Netherlands

8Department of Human Movement Sciences, MOVE Research Institute, Vrije Universiteit, Amsterdam, The

Netherlands

Rationale: Sarcopenia is an emerging clinical challenge in an ageing population and is associated

with serious negative health outcomes. This study aimed to assess the current state of knowledge

about the concept of sarcopenia, diagnostic strategy and management in a cohort of Dutch

healthcare professionals (physicians, physiotherapists, dietitians and others) attending a lecture

cycle on sarcopenia.

Methods: Healthcare professionals (n=223) were asked to complete a questionnaire before, directly

after and five months after attending a lecture cycle on the pathophysiology of sarcopenia,

diagnostic strategy and management of sarcopenia, i.e. interventions and collaboration.

Results: Before attendance, 69.7% of healthcare professionals stated to know the concept of

sarcopenia and 21.4% indicated to know how to diagnose sarcopenia. Handgrip strength was the

most frequently used objective diagnostic measure (33.9%). Five months after attendance, reported

use of diagnostic tests was increased, i.e. handgrip strength up to 67.4%, gait speed up to 72.1%

and muscle mass up to 20.9%. Bottlenecks during implementation of the diagnostic strategy were

experienced by 67.1%; lack of awareness, acquisition of equipment and time constraints to perform

the diagnostic measures were reported most often. Before attendance, 36.4% stated not to consult a

physiotherapists or exercise therapists (PT/ET) or dietitian for sarcopenia interventions, 10.5%

consulted a PT/ET, 32.7% a dietitian and 20.5% both a PT/ET and dietitian. Five months after

attendance, these percentages were 28.3%, 21.7%, 30.0% and 20.0% respectively. A lack of

collaboration was experienced by 36.8%.

Conclusion: The concept of sarcopenia is familiar to most healthcare professionals but application

in practice is hampered, mostly by lack of knowledge, availability of equipment, time constraints and

lack of collaboration.

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(37) Assessment of maximal handgrip strength: how many attempts

are needed?

Esmee M. Reijnierse1, Nynke de Jong2, Marijke C. Trappenburg2,3, Gerard J. Blauw4,5,

Gillian Butler-Browne6, Helena Gapeyeva7, Jean-Yves Hogrel6, Jamie S. McPhee8

Marco V. Narici9, Sarianna Sipilä10, Lauri Stenroth11, Rob C. van Lummel12,13, Mirjam

Pijnappels13, Carel G.M. Meskers13,14, Andrea B. Maier1,13

Affiliations 1Department of Medicine and Aged Care, University of Melbourne, Melbourne, Australia 2Department of Internal Medicine, Section of Gerontology and Geriatrics, VU University Medical Center, Amsterdam, The Netherlands 3Department of Internal Medicine, Amstelland Hospital, Amstelveen, The Netherlands 4Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, The Netherlands 5Department of Geriatrics, Bronovo Hospital, The Hague, Netherlands, 6UPMC UM 76, INSERM U 974, CNRS 7215, Institute de Myologie, Paris, France 7Institute of Sport Sciences and Physiotherapy, University of Tartu, Tartu, Estonia 8School of Healthcare Science, John Dalton Building, Manchester Metropolitan University, Manchester, United Kingdom 9Division of Medical Sciences and Graduate Entry Medicine, MRC-ARUk Centre of Excellence for Musculoskeletal Ageing Research, University of Nottingham, Royal Derby Hospital Centre, Derby, United Kingdom 10Gerontology Research Centre and Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland 11Department of Biology of Physical Activity, University of Jyväskylä, Jyväskylä, Finland 12McRoberts BV, The Hague, The Netherlands 13Department of Human Movement Sciences, MOVE Research Institute, Vrije Universiteit, The Netherlands 14Department of Rehabilitation Medicine, VU University Medical Center, Amsterdam, The Netherlands

Rationale: Handgrip strength (HGS) is used to identify individuals with low muscle strength

(dynapenia). The influence of the number of attempts on maximal HGS is not yet known

and may differ depending on age and health status. This study aimed to assess how many

attempts of HGS are required to obtain maximal HGS.

Methods: Three cohorts (939 individuals) differing in age and health status were included.

HGS was assessed three times and explored as continuous and dichotomous variable.

Paired t-test, intraclass correlation coefficients (ICC) and Bland-Altman analysis were used

to test reproducibility of HGS. The number of individuals with misclassified dynapenia at

attempts 1 and 2 with respect to attempt 3 were assessed.

Results: Results showed the same pattern in all three cohorts. Maximal HGS at attempts 1

and 2 was higher than at attempt 3 on population level (P < 0.001 for all three cohorts). ICC

values between all attempts were above 0.8, indicating moderate to high reproducibility.

Bland-Altman analysis showed that 41.0 to 58.9% of individuals had the highest HGS at

attempt 2 and 12.4 to 37.2% at attempt 3. The percentage of individuals with a maximal

HGS above the gender-specific cut-off value at attempt 3 compared with attempts 1 and 2

ranged from 0 to 50.0%, with a higher percentage of misclassification in middle-aged and

older populations.

Conclusion: Maximal HGS is dependent on the number of attempts, independent of age

and health status. To assess maximal HGS, at least three attempts are needed if HGS is

considered to be a continuous variable. If HGS is considered as a discrete variable to

assess dynapenia, two attempts are sufficient to assess dynapenia in younger populations.

Misclassification should be taken into account in middle-aged and older populations.

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(38) Is Being Malnourished According to the ESPEN Definition for

Malnutrition Associated With Clinically Relevant Outcome Measures

in Geriatric Outpatients?

N.M. van Rijssen RD, MSc, A.G.M. Rojer BSc, M.C. Trappenburg MD, PhD, E.M.

Reijnierse MSc, C.G.M. Meskers MD, PhD, A.B. Maier MD, PhD, M.A.E. de van der

Schueren RD, PhD Submitted

Background and aim: A body of evidence is supporting the association between (the risk

of) malnutrition in relation to physical performance, muscle strength, risk for depression and

cognitive status in geriatric outpatients. Associations between being malnourished

according to the newly proposed ESPEN definition for malnutrition and clinically relevant

outcome measures of the aforementioned variables have not been confirmed yet.

Therefore, the aim of this study was to examine the association between being

malnourished according to the ESPEN definition and clinically relevant outcome measures

in geriatric outpatients.

Methods: Associations between malnutrition and handgrip strength (HGS, kg), Short

Physical Performance Battery (SPPB-score, points), Timed Up and Go test (TUG,

seconds), and Hospital Anxiety and Depression Scale (HADS depression score, points),

were analysed using linear regression. History of falls (falls, yes/no) and a low score on the

Mini Mental-State Examination (MMSE-score ≤24 points) were analysed using logistic

regression. All analyses were adjusted for age and gender.

Results: A total of 185 geriatric outpatients (60% women) with a mean age of 82 (±7.3)

years were included. Being malnourished (8.2%) according to the ESPEN definition was

significantly associated with a lower HGS (-3.38 kg, p=0.031), lower SPPB-score (-1.8

point, p=0.025), higher TUG-time (1.35 times higher time, p=0.020) and higher HADS

depression score (2.03 times higher score, p=0.007). Being malnourished tended towards

an association with falls (OR 3.84, p=0.087). No significant association was found with low

MMSE-score (OR 2.61, p=0.110).

Conclusion: This study is the first to confirm the association between being malnourished,

defined by the ESPEN definition and clinically relevant outcome measures in geriatric

outpatients.

Keywords: malnutrition, nutrition, physical performance, muscle strength, depression,

aged.

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(39) Instrumented assessment of physical activity is associated with

muscle function but not with muscle mass in a general population

A.G.M. Rojer BSc, E.M. Reijnierse MSc, M.C. Trappenburg MD, PhD, R.C. van Lummel MSc, M.

Niessen PhD, K.S. van Schooten PhD, M. Pijnappels PhD, C.G.M. Meskers MD, PhD, A.B. Maier

MD, PhD

Accepted, 26th of june 2017, Journal of Aging and Health.

Objectives: Self-reported physical activity has shown to affect muscle-related

parameters. As self-report is likely biased, this study aimed to assess the association

between instrumented physical activity (I-PA) and muscle-related parameters in a

general population.

Methods: Included were 156 young-to-middle-aged and 80 older community-dwelling

adults. Seven days of trunk accelerometry (Dynaport MoveMonitor, McRoberts B.V.)

quantified daily physical activity (i.e. active/inactive duration, number and mean duration

of active/inactive periods, number of steps per day). Muscle-related parameters

included muscle mass, handgrip strength and gait speed.

Results: I-PA was associated with handgrip strength in young-to-middle-aged adults

and with gait speed in older adults. I-PA was not associated with muscle mass in either

age-group.

Discussion: The association between I-PA and muscle-related parameters was age

dependent. The lack of an association between I-PA and muscle mass indicates the

relevance of muscle function rather than muscle mass.

Keywords: Motor activity; Sarcopenia; Muscle strength; Aged; Activity monitoring;

Physical performance

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(40) Role of glycation on natural killer cell function

Philip Rosenstock1, Kaya Bork1, Vinayaga Srinivasan Gnanapragassam1, Franziska

Frank1, Rüdiger Horstkorte1

1Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany

Natural killer cells (NK cells) are cells of the innate immune system and provide a

major defence against tumor cells and virus infected cells. They are able to kill

modified or infected cells without the need for antigen recognition and can

produce different cytokines to modulate the function of other immune cells. The

activity of NK cells is regulated by activating and inhibitory receptors. In general

NK cells can be divided into two subpopulation based on their expression of

CD56. CD56 is also known as the neural cell adhesion molecule (NCAM) and is a

polysialylated molecule. CD56bright NK cells with a high CD56 expression are

predominantly cytokine producing cells whereas the CD56dim NK cells with a

lower CD56 expression are more cytotoxic. During human aging, which is

associated with an increasing amount of advanced glycation end products

(AGEs) the number, phenotype and function of NK cells is changed. We

investigated the effect of aging on human natural killer cells using the human NK

cell line NK-92 and studied how glycation and sialylation affects the function of

these cells. In our experiments treatment of NK-92 cells with methylglyoxal lead

to glycation and formation of advanced glycation end products. Using a flow

cytometry based cytotoxicity assay we could show that this glycation inhibits the

cytotoxic activity of NK-92 cells.

Presenting author’s email address: [email protected]

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(41) Role of ascorbic acid in protein glycation and PC12

adhesion/neurite outgrowth

Jonas Scheffler1, Kaya Bork1, Vinayaga Srinivasan Gnanapragassam1, Franziska

Frank1, Andreas Simm2, Rüdiger Horstkorte1

1 Institute for Physiological Chemistry, Halle (Saale), Germany

2 Clinic for Heart Surgery, Halle (Saale), Germany

Ascorbic acid is not only known as an important cofactor for multiple enzymatic

pathways including the synthesis of collagen or stresshormones, but also as a major

antioxidant, reducing intra- and extracellular oxidative stress and scavenging free

radicals. However, recent studies suggest an involvement of ascorbic acid and its

degradation products in protein glycation and generation of advanced glycation

endproducts (AGEs). AGEs play an important role in many age-related diseases,

including atherosclerosis, cataract, renal insufficiency and Alzheimer’s disease. Thus,

long-lived proteins like collagens as well as the cells themselves are possibly altered in

stability and function after exposure to high amounts of ascorbic acid, which could lead

to decreased attachment and differentiation. We could show a negative effect of

ascorbic acid-induced glycation on the neurite outgrowth of neuron-like PC12 cells.

Since these cells can adhere on extracellular matrix proteins, we also investigated the

impact of collagen IV-glycation on adhesion and neurite outgrowth.

Presenting author’s email address: [email protected]

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(42) Neural stem and progenitor cells for the in vitro 2D- and 3D-

analysis of human neurogenesis

Jovita Schiller, Matthias Jung, Anne Puls and Dan Rujescu

Neurodegenerative and -developmental diseases such as ASD and schizophrenia are

often associated with genetic variations. Single nucleotide polymorphisms and copy

number variations presumably affect the neural development, resulting in extensive

cognitive impairments that may have a great impact on cell functions. Using the

example of schizophrenia, several risk genes are associated with the disease and some

of them are involved in neural development, neurotransmission as well as the immune

system. Reprogramming of human somatic cells enables the generation of induced

pluripotent stem cells (iPSCs) bearing the genetic information. The neural differentiation

of iPSCs might elucidate the impact of genetic variations and enable the improvement

of antipsychotic drug treatment by sub-classifying patients at a molecular level.

Therefore, patient-specific B cell-derived iPSCs were differentiated into neuroepithelial

rosettes containing self-renewing cells, which were cultured as free-floating

neurospheres to improve cell proliferation. Neurosphere single cells were maintained as

stable populations of neural stem cells (NSCs), which were further differentiated into

functional progenitor and mature neural cells when cultured in an adherent culture

system. Transcript and protein analysis as well as double immunostaining of specific

markers such as SOX2, MSI-1, NEUROG3, and NESTIN confirmed the successful

generation of NSCs as well as more mature neural cells (GFAP, O4, TUBB3, STX, and

NEUN). Patch-clamp recordings verified the presence of different neuron subtypes.

Alternatively, differentiation of neurospheres in a 3D culture system generated cerebral

organoids within 4 weeks, mimicking different brain regions. The regional identity was

confirmed through the expression of telencephalic and hippocampal markers (FOXG1,

OTX1, PROX1, and AUTS2). The presence of early neural cell fates was analyzed

through transcript and protein expression of specific markers as well. Different subtypes

such as GABAergic (GABAAR), glutamatergic (vGLUT1), and motor neurons (HB9)

were also detectable. Together, these results confirmed the efficient neural

differentiation of human iPSCs using both culture systems, enabling functional studies

of healthy and diseased human cortical development concerning the impact of specific

genetic variations.

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(43) PTTG1/securin as a quantitative trait locus candidate gene

controlling progenitor cell survival and organismal lifespan

Desiree Schuetz1, Andreas Brown1, Kalpana J. Nattamai2, Hartmut Geiger1,2

1 Institute of Molecular Medicine, Ulm University, Ulm, Germany

2 EHCB, CCHMC, Cincinnati, USA

Prior work identified a positive correlation between the percentage of hematopoietic

progenitor cells (HPCs), defined as cobblestone-area forming cells (CAFC) day 7, killed

by the genotoxic agent hydroxyurea (HU) and median lifespan in eight inbred mouse

strains. These data suggest a common underlying molecular mechanism for both traits

(response of HPCs to HU and lifespan). Given that HU kills proliferating cells, this

finding was interpreted to mean that the fraction of proliferating HSC/HPCs was

significantly higher in long-lived C57BL/6 (B6) versus short-lived DBA/2 mice. Using

recombinant inbred mice derived from the B6 and DBA/2 mouse strains we mapped a

quantitative trait locus (QTL) on chromosome 11 that is responsible for the HU-killing

phenotype. The locus was found to overlap with a locus that is linked to lifespan, further

emphasizing a likely common mechanism. With this knowledge, congenic mice were

generated that have a B6 background and the specific locus on chromosome 11

replaced by the corresponding segment of DBA/2 mice, named line A, or vice versa for

DBA/2 mice, named line K. Transfer of the loci was verified by single-nucleotide

polymorphism (SNP) analyses. Using the CAFC approach, we could show that HPCs

from DBA/2 and line A mice exhibit higher sensitivity to HU, which confirms that the

locus on chromosome 11 indeed determines the sensitivity of HPCs to HU. We also

analyzed the cell cycle dynamics of HPCs from B6, DBA/2 as well as the congenic

strains. Surprisingly the HPCs of the four mouse strains do not differ in cell cycle

dynamics, which reveals that the distinct response of HPCs to HU is not correlated to

HPCs proliferation. Pituitary tumor-transforming gene 1 (PTTG1, also known as securin)

has been identified as a candidate gene within the locus on chromosome 11, which is

primarily involved in the regulation of sister chromatid separation during cell division.

PTTG1 shows high expression in progenitor cells from DBA/2 and line A mice.

Experiments are underway to verify PTTG1 as the quantitative trait gene in this locus

and to reveal the underlying molecular mechanisms that determine the distinct response

of HPCs to HU treatment.

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(44) High concentrations of methylglyoxal and glyoxal epigenetically

modify histrone H3 in human trophoblast cells in vitro

Tom Seeling1, Katarzyna J. Grybel1, S. Mareike Pendzialek1, Maria Schindler1, Axel

Imhof2, Ignasi Forné2 and Anne Navarrete Santos1

1Department of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg,

Faculty of Medicine, Halle (Saale), Germany 2Department of Medical Biology, Ludwig Maximilians University Munich, Faculty of

Medicine, Munich, Germany

The Developmental Origins of Health and Disease (DOHaD) hypothesis states, that

long-term health and onset of ageing-associated diseases are already set during fetal

development. Particularly, the maternal metabolism influences the embryonic uterine

environment with consequences for trophoblast and placental development in early

pregnancy. In western society the first-birth age for women increases, leading in part to

higher risk of suffering from metabolic diseases like diabetes mellitus. Diabetes mellitus

is characterized by high concentrations of glucose and its metabolites like methylglyoxal

(MGO) and glyoxal (GO).

The aim of this study was to analyze the influence of a diabetic environment associated

with high levels of MGO and GO on histone protein modifications and histone-modifying

enzymes in placental cells.

Histone modifications were examined by LC-MS/MS. Therefore, the human trophoblast

cell line AC-1M88 was cultured with 100 µM MGO and GO simultaneously for 72 hours.

The exposition to MGO and GO changed the methylation pattern on several lysines,

especially on K9 and K27. Higher methylation was also detected at K79 after exposition

to MGO and GO, which is a marker for aging cells. Additionally expression of

methyltransferases and demethylase were examined on transcriptional level.

Our data show that trophoblast cells challenged by aging factors (MGO and GO),

changed epigenetic background to support survival through alterations in histone

modifications. These changes may promote metabolic programming with consequences

in later life.

This work was supported by the German Research Council (DFG RTG ProMoAge 2155), the FP7-

EpiHealthNet (N°317146), COST Action EpiConcept FA 1201 and SALAAM BM1308.

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(45) Lysine acetylation patterns in T-Cells in the context of ageing

Georgiana Toma, Dagmar Quandt and Barbara Seliger

Institute of Medical Immunology, Martin-Luther University Halle, Germany

Ageing is a complex process affecting a wide range of physiological functions, including

the immune system, for which the age-related alterations are called

immunosenescence. One factor contributing to this process is T cell senescence.

Posttranslational modifications (PTM) of proteins are one of the key mechanisms

involved in aging. The most abundant PTMs are phosphorylation and acetylation.

Protein Acetylation is regulated by 2 types of enzymes: acetyltranferases and

deacetylases (initially named histone deacetylases – HDAC). HDACs are grouped into

four classes, the third being Sirtuins, and they have various substrates beyond histones.

Sirtuins, in particular Sirtuin 1, 3 and 6 have been associated to ageing. Increasing the

functionality of sirtuin-1 has been proven to prolong lifespan in certain model systems.

How T cell senescence is regulated by alterations of acetylation of key transcription

factors as well as cellular proteins is currently unknown.

Therefore our aim is to identify age-related alterations within the acetylation pattern of

different T cell subsets. This analysis will include acetyltransferase as well as HDAC

expression studies on mRNA and at the protein level. In addition we will analyse the

enzymatic activity of those enzymes in T cells.

Our current strategy is to determine differences in the protein acetylation patterns of

CD8+ and CD4+ T cells sorted from PBMCs obtained from young (< 30 years old) and

older (> 60 years old) healthy blood donors. To visualize the acetylation patterns, we

treat the cells with trichostatin a (TSA) – a HDAC class I and II inhibitor and

nicotinamide (NAM) – an inhibitor of HDAC class III (SIRT). Preliminary results show

more dramatic alterations in T cell acetylation patterns upon TSA treatment. That would

speak for a more prominent role of HDAC I and II as compared to sirtuins. Differentially

acetylatetd candidate proteins will be identified by mass spectrometry and functional T

cell studies will be performed.

Results of our study will potentially help to find new ways to improve and modulate T

cell immunity of the elderly.

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(46) Epigenetic regulation of gene transcription by non-enzymatic

protein modification

Arina Urazova1, Kristin Wächter1, Patrick Winterhalter1, Wolfgang Hoehenwarter2,

Jochen Balbach3, Andreas Simm1

1University Hospital Halle, Clinic for Heart Surgery, Halle (Saale)

2Leibniz Institute of Plant Biochemistry, Halle (Saale)

3Institute of Physics, Halle (Saale)

E-mail: [email protected]

Formation of Advanced Glycated End Products (AGEs) is a result of glycation, which is

non-enzymatic reaction between reducing sugars, or other α-dicarbonyl compounds,

and amino groups of proteins, lipids and nucleic acids. AGEs accumulate during aging

and have been implicated in the pathophysiology of numerous age-related diseases.

Glycation of nuclear proteins at lysins or arginins may alter their function and may have

an impact on epigenetic regulation of gene transcription.

Glycated nuclear proteins from young and replicative senescent cells, as well as from

normal and tumor cells, are going to be compared, and identified modified proteins will

be further analyzed.

Nuclei from human embryonic kidney cells 293A (HEK293A) and human kidney

carcinoma cell line Caki-2 were isolated and AGE-modified nuclear proteins were

compared via western blot using antibody against carboxymethyl-lysine (CML),

hydroimidazolone MG-H1 and argpyrimidine. Different pattern of modified proteins in

HEK293A and Caki-2 cell lines was observed. LC-MS/MS analysis revealed that in

HEK293A cells 7 out of 3424 identified proteins are modified, while in Caki-2 cells

among 2701 proteins 11 are modified. Two of them, Histone H4 and Myb-binding

protein 1A, were identified in both cell lines. Amount of modified peptides (12 out of

32127 identified peptides in HEK293A cells and 14 out of 23528 in Caki-2 cells) is too

low for reproducible quantification of differences in these modifications.

Immunoprecipitation was used to enrich samples with modified proteins prior to

analysis, enrichment steps are elucidated and optimized.

Identified AGE-modification of lysins/arginins within nuclear proteins indicate on a

possibility to modify the epigenetic regulation of cells.

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(47) Only Body Mass Index prredicts metabolic syndrome in older

people

Jeanine M. Van Ancum MD a, Nini H. Jonkman PhD a, Natasja M. van Schoor PhD b,

Emily Tressel Bsc a, Carel G.M. Meskers MD, PhD c, Mirjam Pijnappels PhD a, Andrea

B. Maier MD, PhD a,d

a Department of Human Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, The

Netherlands. b

Amsterdam Public Health Research Institute, Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands

c Department of Rehabilitation Medicine, VU University Medical Center, Amsterdam Movement Sciences, the

Netherlands. d

Department of Medicine and Aged Care, Royal Melbourne Hospital, University of Melbourne, Melbourne, Victoria,

Australia.

Background: Demographics, lifestyle, clinical measures and physical performance

have individually been shown to be associated with metabolic syndrome (MetS).

However, the combination of these variables has not yet been investigated in a large

population of older people. We aimed to identify independent determinants of MetS

prevalence, as well as predictors of MetS at three year follow-up in older people.

Methods: Demographics, lifestyle, clinical measures and physical performance, along

with the prevalence and incidence of MetS over three years were collected within the

Longitudinal Aging Study Amsterdam including individuals aged 55 years and older.

MetS was defined according to the National Cholesterol Education Program Adult

Treatment Panel III (NCEP-ATPIII). Stepwise backward selection in logistic regression

analyses was used to identify independent determinants of MetS prevalence in a cross-

sectional analysis (n=1292) and predictors of MetS incidence at three year follow-up

(n=218).

Results: MetS was prevalent in 37.1% individuals (n=479), and MetS incidence over

three years was 30.3% (n=66). In the cross-sectional analysis, heart disease, artery

disease, diabetes, alcohol consumption and Body Mass Index (BMI) were significantly

associated with MetS. In the longitudinal analysis, only BMI was an independent

predictor of MetS.

Conclusion: BMI predicts the development of MetS over three years, providing a first

step towards an easy-to-assess screening tool for healthcare in older people.

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(48) Muscle strength and muscle mass in older patients during

hospitalization: the EMPOWER study

Jeanine M. Van Ancum MD a, Kira Scheerman MSc

b, Vincent D. Pierik BSc

b, Siger T.

Numans BSc b, Sjors Verlaan MSc

b, Hanne E. Smeenk MD

c, Monique Slee-Valentijn MD

d,

Roeliene C. Kruizinga MD, PhD b, Carel G.M. Meskers MD, PhD

e, Andrea B. Maier MD,

PhD a,f

a Department of Human Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, The

Netherlands b Department of Internal Medicine, Section of Gerontology and Geriatrics, VU University Medical Center, Amsterdam

Movement Sciences, the Netherlands. c GERION, Department of General Practice and Elderly Care Medicine, VU University Medical Center, Amsterdam,

the Netherlands. d Center of Excellence in Geriatric Rehabilitation, Cordaan, Amsterdam, the Netherlands.

e Department of Rehabilitation Medicine, VU University Medical Center, Amsterdam Movement Sciences, The

Netherlands f Department of Medicine and Aged Care, Royal Melbourne Hospital, University of Melbourne, Melbourne, Victoria, Australia.

Background: Low muscle strength and muscle mass are associated with an increased length

of hospital stay and higher mortality rate in inpatients. To what extent hospitalization affects

muscle strength and muscle mass is unclear. We aimed to assess muscle strength and muscle

mass at admission and during hospitalization in older patients and their relation with being at

risk of four geriatric conditions: delirium, falls, malnutrition and functional disability.

Methods: The EMPOWER inception cohort included patients aged 70 years and older,

admitted to four wards of the VU university medical center in the Netherlands between April and

December 2015. At admission, patients were screened for being at risk of four geriatric

conditions. Both at admission and at discharge, muscle strength and muscle mass were

assessed.

Results: A total of 373 patients (mean age, SD: 79.6, 6.38 years) were included at admission of

which 224 patients (mean age, SD: 80.1, 6.32 years) were left for analysis at discharge. At

admission, lower muscle strength in both female and male patients and low muscle mass in

male patients were associated with being at risk of a higher cumulative number of geriatric

conditions. Muscle strength increased during hospitalization, but no change in muscle mass

was observed. Changes in muscle measures were not associated with being at risk of geriatric

conditions.

Conclusion: Older patients with lower muscle strength and muscle mass at admission were at

risk of a higher cumulative number of geriatric conditions. However, being at risk of geriatric

conditions did not forecast further decrease in muscle strength and muscle mass during

hospitalization.

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(49) Muscle strength and muscle mass are associated with pre- and

posthospitalization falls in older male patients

Jeanine M. Van Ancum MD a, Mirjam Pijnappels PhD a, Nini H. Jonkman PhD a, Kira

Scheerman MSc b, Sjors Verlaan MSc b, Carel G.M. Meskers MD, PhD c, Andrea B.

Maier MD, PhD a,d

a Department of Human Movement Sciences, Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, The

Netherlands. b

Department of Internal Medicine, Section of Gerontology and Geriatrics, VU University Medical Center, Amsterdam Movement Sciences, the Netherlands. c Department of Rehabilitation Medicine, VU University Medical Center, Amsterdam Movement Sciences, the

Netherlands. d

Department of Medicine and Aged Care, Royal Melbourne Hospital, University of Melbourne, Melbourne, Victoria, Australia.

Introduction: Low muscle strength and muscle mass are associated with falls in

community-dwelling older adults, but it is unknown if these muscle measures are also

associated with falls in older inpatients. This study aimed to investigate the association

between muscle measures and pre- and post-hospitalization falls in older inpatients.

Methods: An inception cohort of inpatients aged 70 years and older admitted to an

academic teaching hospital were included in this study, from April to December 2015.

Handgrip strength (HGS) and muscle mass were measured at admission using

dynamometry and bioelectrical impedance analysis. Pre-hospitalization falls were assessed

at admission with a questionnaire and dichotomized as at least one fall in six months before

admission. Post-hospitalization falls were assessed by telephone interview three months

after discharge and dichotomized as at least one fall. Associations were analyzed with

logistic regression analysis, stratified for gender.

Results: The study included 378 inpatients (mean age, SD: 79.7, 6.4). Pre-hospitalization,

50% of female and 41% of male patients experienced at least one fall. Post-hospitalization,

18% of female and 23% of male patients experienced at least one fall. Lower HGS was

associated with both pre- and post-hospitalization falls, and lower muscle mass was

associated with post-hospitalization falls in male, but not in female patients.

Conclusion: These findings highlight the likely involvement of muscle strength and muscle

mass in the occurrence of pre- and post-hospitalization falls in males, indicating a possible

target for interventions in older male patients with low HGS and muscle mass upon

admission to the hospital.

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(50) Mitochondria preconditioning to promote healthy aging in

C.elegans

Natascia Ventura

The nematode C.elegans is a powerful model organism for aging studies. Hormesis or

preconditioning is a phenomenon by which the body benefits from various kinds of

moderate stress, which at high doses are harmful. A growing body of evidence indicates

that different forms of preconditioning could be exploited as a strategy to protect against

degenerative disorders in human and to extend lifespan in C. elegans.

Mitochondria play a central role in the aging process and hormetic-like effects on

C. elegans lifespan have been observed following different degrees of mitochondrial

stress [1, 2]. In the past years we have shown that either genetic or pharmacological

interventions reducing mitochondrial activity can promote C. elegans healthspan [3, 4].

Moreover we have identified different evolutionarily conserved, cytoprotective

responses causally involved in mitochondrial stress control of longevity such as

autophagy or transcription factors (p53 and Hif1) [2, 5]. Thanks to a recently established

phenotypebased screening platform [6] we are currently trying to identify possible

dietary components promoting healthspan through mitochondrial preconditioning.

1. Rea, S.L.*, Ventura N.*, and Johnson T.E. Relationship between mitochondrial electron transport chain dysfunction, development, and life extension in Caenorhabditis elegans. PLoS Biol, 2007. 5(10): p. e259. 2. Ventura, N., et al., p53/CEP-1 increases or decreases lifespan, depending on level of mitochondrial bioenergetic stress. Aging Cell, 2009. 8(4): p. 380-93. 3. Maglioni, S., et al., Mitochondrial stress extends lifespan in C. elegans through neuronal hormesis. Exp Gerontol, 2014. 56: p. 89-98. 4. Schiavi, A., et al., Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans. Exp Gerontol, 2013. 48(2): p. 191-201. 5. Schiavi, A., et al., Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans. Curr Biol, 2015. 25(14): p. 1810-22. 6. Maglioni, S., et al., An automated phenotype-based microscopy screen to identify pro-longevity interventions

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(51) Isolation and characterization of adipose-derived stem cells

(ASCs) from young and old female rabbits

Christin Volk1, Alexander Navarrete Santos2, Matthias Jung3, Anne Navarrete Santos1,

and Juliane-Susanne Jung1

1 Department of Anatomy and Cell Biology,

2 Centre for Medical Basic Research,

3 Department of Psychiatry, Psychotherapy, Psychosomatic Medicine, all: Martin Luther University Halle-Wittenberg,

Halle (Saale), Germany

The UN estimates that the number of people older than 65 years will grow from 7% in 2000 to 16%

in 2050 worldwide. Therefore, research on aging mechanisms and aged-related disease is one

major issue of our ageing world. Adult stem cells such as mesenchymal stem cells are affected by

molecular and cellular aging mechanisms. Mesenchymal stem cells have the capability to self-renew

and can differentiate into multiple cell types of the mesoderm germ layer. Bone marrow-derived stem

cells represent a well-established model, but donor material is limited and requires invasive

methods. Adipose tissue is an alternative source of mesenchymal stem cells due to facile harvesting

techniques. Therefore adipose-derived stem cells (ASCs) represent a promising tool for studying the

biological characteristics of tissue regeneration and age-related diseases in detail.

In the present study, ASCs were isolated from visceral adipose tissue of young (16 weeks) and old

female rabbits (>108 weeks). ASCs were prepared from tissue by mincing and dissociation with

collagenase A. ASCs were cultured under appropriate cell culture conditions until passage 3. Cells

were analyzed morphologically by microscopy at day 7, 10, 15, and 21. Furthermore, flow cytometry

and transcript analysis were used for molecular characterization of ASCs and differentiated early

adipocytes.

After 10 days of culturing, in vitro ASCs showed the typical morphology of mesenchymal stem cells.

Most of the cells were positive for the ASCs-associated cluster of differentiation (CD) 105 (94.3%)

and CD73 (51.2%) and negative for CD14 (0.0%), demonstrating their mesenchymal origin. Isolated

ASCs expressed mesenchymal stem cell markers including Myc, Klf4, Chd1, Rest and Kat6A, while

the pluripotency-associated genes like Nanog, Oct4 and Sox2 were not expressed. Adipogenic

differentiation of the ASCs (passage 3) was induced by dexamethasone (1.0 μM final), 3-isobutyl-1-

methylxanthine (IBMX - 0.5 mM final), indomethacin (0.2 mM final) and insulin (10.0 μM final)

enabling the analysis of aging during fat cell generation and regeneration. The adipogenic

differentiation efficiency was low, with 4.3% for ASCs from young rabbits. In a next step the ASC

differentiation procedure will be optimized to increase adipogenic differentiation.

Together, we successfully established rabbit ASC cultures representing an in vitro model for the

analysis of stem cell aging mechanisms.

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(52) Bakery products as functional food

Kristin Wächter1, Alexander Navarrete Santos2, Veronika Somoza3, Hendrik Treede1,

Andreas Simm1, 2,

1 Clinic for Heart Surgery, Martin-Luther-University Halle-Wittenberg, Halle (Saale)

2 Center for medical basic research, Martin-Luther University Halle-Wittenberg, Halle (Saale)

3 Research Platform Molecular Food Science, University of Vienna, Vienna, Austria

Advanced glycation end products (AGEs) are the result of a non-enzymatic reaction of

proteins with reactive carbohydrates. Heat-processed food like bread contains high

amounts of AGEs. Numerous previous studies suggest an antioxidative and

cardioprotective potential of extracts from the bread crust (BCE). The activation of NF-

κB signaling pathway by BCE is well understood. However it is largely unknown whether

NRF2, the master regulator of oxidative stress resistance in mammalian cells, is

affected by BCE. Furthermore to date there is only little knowledge concerning the

bioactive compounds, which accounts for the antioxidant BCE-properties.

Understanding the molecular mechanism of BCE action is an important prerequisite to

establish BCE as a functional food product. Therefore, we developed a cell culture

model to elucidate the effect of different bread crust extracts on NF-κB as well as NRF2

activation in an endothelial reporter cell line (EAhy926 dual plasmid, NF-κB–GFP,

NRF2-mCherry). An increased nuclear translocation of NF-κB and NRF2 in these cells

results in elevated GFP and mCherry expression, which are analyzed by flow cytometry.

Our data showed for the first time, that soluble extracts from bread crust are capable to

stimulate not only NF-κB but also NRF2 signaling pathways in endothelial cells in a

dosage- and time-dependent manner. In a second approach, we uncovered that AGE

modified BSA is able to activate both transcription factors in comparison to unmodified

BSA in endothelial cells. This tempts us to speculate, that especially AGE-modified

proteins present in BCE could mediate the antioxidative potential. In addition, by size

exclusion experiments we found that peptides smaller than 3 kDa are sufficient for

NRF2 and NF-κB activation.

In continuative studies we want to optimize and enhance the action of BCE by changing

the baking conditions and ingredients. Furthermore, the results will be confirmed by

feeding studies in mice.

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(53) Development of a tBHP-induced senescence model to study

molecular mechanisms of extrinsic skin aging

Sophia Wedel1, Maria Cavinato1, Pidder Jansen-Dürr1

1 Institute for Biomedical Aging Research, Innsbruck University, Innsbruck, Austria

Skin aging is influenced by intrinsic and environmental factors. Epidemiological studies

and many research groups have shown that sunlight, tobacco smoke and air pollution

can prematurely induce aging in skin. Still, the mechanisms that underlie the changes of

the skin’s integrity and appearance upon exposition to these extrinsic factors are

scarcely understood. Therefore, it is of great importance to develop new model that help

us to improve our understanding of this matter.

We have established a model for studying the mechanisms of extrinsic skin aging by

treating human dermal fibroblasts and reconstructed human skin equivalents with eight

sublethal doses of the organic peroxide tert-butyl-hydroperoxide (tBHP). The oxidative

stress injury and depletion of antioxidant defense resulting from tBHP treatment are

very similar to and consequently able to mimic the effects of tobacco smoke and

particulate matter on skin.

We found that tBHP treatment increased intracellular reactive oxygen species (ROS)

levels and subsequently induced DNA damage response in HFFs. Additionally, we were

able to show that tBHP induced changes in morphology and led to proliferative arrest by

triggering regulation of proteins that are important for cell cycle progression and stress

response including p53, p21cip/waf and pRB, p16INK4A in the fibroblast monolayer.

Furthermore, more than 90% of treated HFFs were positive for the activity of

senescence-associated β-galactosidase nine days after the tBHP administration

started. At this point in time cell death occurred infrequently. In human skin equivalents

treatment with tBHP increased thickness of stratum corneum and the abundance of

pyknotic nuclei in the epidermis, which can also be detected upon UV irradiation.

We have developed a new model system for extrinsic skin aging, employing treatment

with tBHP and inducing a senescent phenotype in fibroblasts. Additionally, we were able

to collect preliminary on skin equivalents. This new experimental setup allows us to do

further research on mechanisms of extrinsic skin aging and ultimately helps to improve

existing and map out novel anti-skin aging strategies.

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(54) Determination of AGE modifications: To detect or not to detect,

that is the question

Patrick Rainer Winterhalter, Andreas Simm

BACKGROUND: In human age-related disorders, the in vivo formation of advanced

glycation end products (AGEs) is an important and well-known pathogenic event. The

generation of AGEs under pathological conditions is an unpreventable process via the

slow, non-enzymatic formation of amino acids with omnipresent metabolites of the

glycolysis as carbohydrates or dicarbonyls. However, the detection of AGEs is still

problematic and sometimes non-reproducible. Reasons might be the high variety of

possible modifications that are often not identified yet. In addition, the low abundance

and the insufficient identification tools hamper the description of AGEs and associated

dysfunctions.

OBJECTIVE: To determine the enrichment and detection conditions for AGE

modifications, in particular several mono- and polyclonal anti-AGE antibodies were

tested by protein immunoblot.

METHODS: Anti-AGE antibodies from different sources were analyzed utilizing non- or

exogenously AGE-modified model proteins as well as crude AKR-2B cell line lysate.

Thereby, the affinity and specificity for AGEs in general and for specific AGEs as well as

the test conditions were analyzed. A green fluorescent secondary antibody visualized

the protein immunoblots.

RESULTS: For monoclonal anti-AGE antibodies, the sensitivity of the specific AGE

detection is mainly limited. Further, an antigenic effect of surrounding amino acids is

assumed. In addition, the blocking solutions mainly modify the results. Polyclonal

antibodies have clear benefits regarding sensitivity and specificity.

CONCLUSION: A successful determination of AGEs mainly depends on the used

antibody and conditions.

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(55) Glycation of Wnt inhibits canonical signaling

Zhennan Ye1, Sonnhild Mittag1, Andreas Simm2, Otmar Huber1

1Institute of Biochemistry II, Jena University Hospital, Friedrich Schiller University Jena, Nonnenplan 2-4, D-07743

Jena, Germany; 2Department of Cadiothoracic Surgery, University Hospital Halle, Ernst-Grube Str. 40, D-06120 Halle (Saale),

Germany

Signaling pathways involved in repair, regeneration and stem cell proliferation often

exhibit reduced activity during aging. Accumulation of advanced glycation endproducts

(AGEs) is a typical sign of aging tissues and organs. We here addressed if this

posttranslational modification affects Wnt/β-catenin signaling. Glyoxal (GO) or

methylglyoxal (MGO) treatment of Wnt3a-conditioned medium inducing glycation of

medium components reduces Topflash reporter gene activity. Control experiments

provide evidence that glycation of Wnt3a and not of other medium components are

responsible for this effect. In this respect we show a GO-induced molecular weight shift

of immunoprecipitated Wnt3a. In our hands it appears that AGEs per se can stimulate

canonical Wnt signaling, whereas glycation of Wnt has an inhibitory effect. Consistent

with lowered reporter gene activity, reduced transcription of endogenous target genes

and modulation of total β-catenin and phospho-β-catenin levels were observed. From

these results we speculate that the final effects of glycation depends on an interplay of

RAGE- and Wnt-mediated effects.

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List of Speakers A – G Campisi, Judith Buck Institute for Research on Aging Novato, 8001 redwood Boulevard CA 94945, USA Lawrence Berkeley National Laboratory, Berkeley CA 94720, USA E-Mail: [email protected] [email protected] [email protected] Tel.: +1 (0) 415 - 209 2043

Chondrogianni, Niki Institute of Biology, Medicinal Chemistry and Biotechnology 48 Vassileos Constantinou Ave. 116 35 Athens, GR E-mail: [email protected] Tel.: +30 (0) 210-7273768 Fax: +30 (0) 210-7273677

de Grey, Aubrey Aubrey de Grey SENS Research Foundation 110 Pioneer Way, Suite J Mountain View, CA 94041, USA E-Mail: [email protected] Tel: +1 650 938 6100

Eifert, Sandra Klinikum der Universität München Campus Grosshadern Herzchirurgische Klinik und Poliklinik Marchioninistraße 15 81377 München, D E-Mail: [email protected] Tel.: +49 (0) 89 – 4400 73458 Fax: +49 (0) 89 – 4400 78898

Emmert, Maximilian UniversitätsSital Zürich Universitäres Herzzentrum Zürich Rämistraße 11 8091 Zürich, CH E-Mail: [email protected] Tel.: +41 (0) 44 – 255 9362

Franceschi, Claudio University of Bologna Department of Experimental, Diagnostic and Specialty Medicine DIMES Via Zamboni, 33 40126 Bologna BO, I E-Mail: [email protected] Tel.:

Fülöp, Tamas Université de Sherbrooke Département de pédiatrie /Dienst d’immunologie-allergologie 2500, boul. Kanada J1K 2R1, CAN E-Mail: [email protected] Tel.: +1 (0) 819 – 821 8000

Gekle, Michael Julius-Bernstein-Institut für Physiologie Magdeburger Straße 6 D-06112 Halle (Saale) E-Mail: [email protected]

Tel.: +49 (0) 345 – 557 1389

Fax: +49 (0) 345 – 557 4019

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List of Speakers G - L

Gosch, Markus Universitätsprofessor der Paracelsus Medizinischen Privatuniversität für Geriatrie Chefarzt Med. Klinik 2 - Geriatrie Klinikum Nord Prof.-Ernst-Nathan-Straße 1, 90419 Nürnberg, D E-Mail: [email protected] Tel.: +49 (0) 911 398 - 2435 Fax: +49 (0) 911 398 - 2117

Ground, Ian Richard Department of Philosophy University of Hertfordshire Hatfield AL10 9AB, UK E-Mail: [email protected]

Grune, Tilman Deutsches Institut für Ernährungsforschung Potsdam-Rehbrücke Arthur-Scheunert-Allee 114-116 14558 Nuthetal, D Assistentin: Stephanie Wirth E-Mail: [email protected] Tel. +49 (0) 33200 - 88-2416 Fax +49 (0) 33200 - 88-2555

Haendeler, Judith IUF – Leibniz-Institut für umwelt-medizinische Forschung gGmbH Auf’m Hennekamp 50 40225 Düsseldorf, D

E-Mail: [email protected]

Tel.: +49 (0) 211 3389 291

Hoeijmakers, Jan Erasmus MC University Medical Center Rotterdam P.O. Box 2040, 3000 CA Rotterdam, NL

E-Mail: [email protected] Tel.: +31 (0) 10 – 7044844

Kirkland, James Robert and Arlene Kogod Center on Aging Minnesota-Rochester Mayo-Klinik 200 Erste St. SW Rochester, MN 55905, USA E-Mail: [email protected] Kari Sczepanski Administrative Assistant Email: [email protected] Phone: +1 (0) 507 266-9151 Fax: +1 (0) 507 293-3853

Klöss, Thomas Martin-Luther-Universität Halle-Wittenberg Ärztlicher Direktor Ernst-Grube-Straße 30 06120 Halle (Saale), D

E-Mail : [email protected] Tel. : +49 (0) 345 – 557 4481

Lepperdinger, Günter Universität Salzburg Hellbrunnerstraße 34 5020 Salzburg, AT

E-Mail: [email protected] Tel.: +43 (0) 662 - 8044 5739 Fax: +43 (0) 662 - 8044 144

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List of Speakers M - R

Maier, Andrea The University of Melbourne Melbourne Health The Royal Melbourne Hospital - City Campus, Level 6 North Grattan Street Parkville Victoria 3052, AUS E-Mail: [email protected] Tel.: +61 (0) 3 - 8387 2137 Fax: +61 (0) 3 - 8387 222

Martin, George M. University of Washington Department of Pathology Professor of Pathology Emeritus K-543 Health Sciences Building BOX 357470 1959 NE Pacific Street Seattle, WA 98195-7470, USA E-Mail: [email protected] Tel.: +1 (0) 206 - 543 5088 Fax: +1 (0) 206 - 685 8356

Mirelman, Anat Director- Laboratory of Early Markers of Neurodegeneration (LEMON) Department of Neurology Tel Aviv Sourasky Medical Center 6 Weizmann st, Tel Aviv 64239, IL

E-Mail: [email protected] Tel: +972 (0) 3 6973960 Fax: +972 (0) 3 6947514

Moskalev, Alexey Syktyvkar State University; Institute of Biology, Komi Science Center of the Ural Division of the Russian Academy of Science; MIPT (State University) 28 Kommunisticheskaja Str. 167982 Syktyvkar, RUS E-Mail: [email protected] Tel.: +7 (0) 8212312894

Munoz-Canove, Pura ICREA and Pompeu Fabra University Department of Experimental and Health Sciences Dr. Aiguader, 88 08003 Barcelona, ES E-Mail: [email protected]

Niemann, Bernd Klinik für Herz-, Kinderherz- und Gefäßchirurgie Universitätsklinikum Giessen und Marburg Standort Giessen Rudolf Buchheim Strasse 7 35392 Giessen, D E-Mail: [email protected]

Tel.: +49 (0) 641 - 98556233 Fax: +49 (0) 641 - 98544309

Rheinsberg, Zoe

Universität Tübingen Institut für Ethik und Geschichte der Medizin Gartenstr. 47

72074 Tübingen, D E-Mail : [email protected] Tel. : +49 (0) 7071 – 2978032

Rudolph, Lenhard Fli Leibniz-Institut für Alternsforschung Fritz-Lipmann-Institut e.V Beutenbergstraße 11 07745 Jena, D

E-Mail: [email protected]

Tel.: +49 (0) 3641 – 656000 Sekretariat

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List of Speakers S - T

Sebekova, Katarina Institute of Molecular Biomedicine Medical Faculty Comenius University Sasinkova 4 811 08 Bratislava Slovakia E-Mail: [email protected] Tel.: +421 (0) 2 59357 429 mobile: +421 (0) 907 794 093

Simm, Andreas Universitätsklinik und Poliklinik für Herzchirurgie Ernst-Grube-Str. 40 D-06120 Halle (Saale) E-Mail: [email protected] Tel.: +49 (0) 345 – 557 2647 Fax: +49 (0) 345 – 557 7070

Treede, Hendrik

Universitätsklinik und Poliklinik für Herzchirurgie

Ernst-Grube-Straße 40 D-06120 Halle (Saale) E-Mail : [email protected] Tel. : +49 (0) 345 – 557 2720

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Organizers

Treede, Hendrik Department of Cardiac Surgery Ernst-Grube-Straße 40 D-06120 Halle (Saale) E-Mail: [email protected] Tel.: +49 (0) 345 – 557 2720 Fax: +49 (0) 345 – 557 2782

Simm, Andreas Department of Cardiac Surgery Ernst-Grube-Str. 40 D-06120 Halle (Saale) E-Mail: [email protected] Tel.: +49 (0) 345 – 557 2647 Fax: +49 (0) 345 – 557 7070

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Involved Partners

German National Academy of Sciences Leopoldina Jägerberg 1 D-06108 Halle (Saale) E-Mail: [email protected] Tel.: +49 (0) 345 - 47239 600 Fax: +49 (0) 345 - 47239 919 DGGG German Society of Gerontology and Geriatrics Geschäftsstelle Seumestr. 8 D-10245 Berlin E-Mail: [email protected] Tel.: +49 (0) 30 - 52137 271 Fax: +49 (0) 30 - 52137 372

ProMoAge Martin-Luther-Universität Medizinische Fakultät Herzchirurgie Ernst-Grube-Str. 40 06120 Halle Telefon: +49 345/557-3041

IZAH Interdisciplinary Centre on Ageing Halle Ernst-Grube-Str. 40 D-06120 Halle E-Mail: [email protected] Tel.: +49 (0) 345 - 557 2647 Fax: +49 (0) 345 - 557 7070

Friends and Sponsors of the Martin-Luther-University Halle-Wittenberg e.V. Universitätsplatz 10 D-06108 Halle E-Mail: [email protected] Tel.: +49 (0) 345 - 552 2912 Fax: +49 (0) 345 - 552 7076

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Author Index Ale-Agha, N. 59, 60

Altschmied, Y. 59, 60, 62, 73

Amoah, A. 61

Andreas, L. 87

Antebi, A. 90

Aufenvenne, K. 62, 73

Balbach, J. 104

Baldensperger, T. 63, 80

Barthel, F. F. 75

Bertinat, R. 70

Beyer, A. 90

Bezold, V. 64

Bilova, T. 86

Birkemeyer, C. 86

Blauw, G. J. 95

Bork, K. 64, 68, 98, 99

Brandt, W. 86

Brauch, D. 86

Brown, A. 101

Butler-Browne, G. 95

Cabbia, A. 65

Campisi, J. 54

Cavinato, M. 66, 111

Chondrogianni, N. 22

Cifuentes, M. 70

de Grey, A. 38

de Jong, N. 95

de van der Schueren, M. A. E. 93, 94, 96

Demuth, H.-U. 18

Di Sanzo, S. 67

Ding, J. 85

Doves, M. 94

Eberlein, P. 75

Ehrhardt, T. 64, 68, 74, 79

Eifert, S. 32

Emini, R. 61

Emmert, M. 46

Ender, C. 69

Florian, C. 61

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Forman, K. 70

Forné, I. 102

Franceschi, C. 8

Frank, F. 98, 99

Frolov, A. 86

Fülöp, T. 16

Gapeyeva, H. 95

Gebert, N. 89

Geiger, H. 61, 101

Giegling, I. 68, 74, 79

Glaubitz, N. 88

Glomb, M. A. 63, 76, 80

Gnanapragassam, V.S. 72, 98, 99

Gosch, M. 28

Goy, Ch. 60, 62, 73

Grabe, S. 71

Großmann, C. 78

Ground, I. R. 36

Grune, T. 20, 69

Grybel, K. J. 102

Haendeler, J. 30, 59, 60, 62, 73

Hartmann, C. 68, 74, 79

Hause, G. 85

Haybäck, J. 87

Heinemann, S. H. 82

Heiss, Ch. 62

Heller, R. 69

Henning, C. 76

Hermann, M. 66

Hoehenwarter, W. 104

Hoeijmakers, J. 56

Hofmann, B. 75, 88

Hogrel, J.-Y. 95

Hohmann, C. 76

Hollemann, Th. 85

Horstkorte, R. 64, 72, 75, 88, 98, 99

Huber, N. 77

Huber, O. 113

Hübschmann, R. 78

Hönicka, M. 61

Ignatov, A. 87

Imhof, A. 102

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Jacobs, K. 88

Jakobs, Ph. 73

Jansen-Dürr, P. 66, 111

Jasper, H. 89

Jenewein, B. 66

Jonkman, N. H. 105, 107

Jung, J.-S. 71, 109

Jung, M. 64, 74, 79, 100, 109

Junk, A. 80

Kaether, Ch. 81

Kalinski, Th. 87

Keller, A. 61

Kirkland, J. 52

Kirkpatrick, J. 89

Klotz, L.-O. 81

Korzelius, J. 89

Koziel, R. 66

Kruizinga, R. C. 91

Kryeziu, N. 69

Kuldyushev, N. A. 82

Kummer, A. 80, 83

Köhnlein, K. 81

Langner, J. 84

Lepperdinger, G. 48

Liebold, A. 61

Liehr, K. 76

Liu, H. 85

Maier, A. 10, 91, 93, 94, 95, 96, 97, 105, 106, 107

Mamontova, T. 86

Martin, G. M. 34

Martinez, F. 70

McPhee, J. S. 95

Mece, O. 69

Meskers, C. G. M. 91, 93, 94, 95, 96, 97, 105, 106, 107

Milrud, S. 86

Mirelman, A. 14

Mittag, S. 113

Moskalev, A. 50

Munoz-Cánove, P. 44

Nagasundaram, M. 72

Narici, M. V. 95

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Naß, N. 87

Nattamai, K. J. 101

Navarrete-Santos, Alex 71, 83, 88, 92, 109, 110

Navarrete-Santos, Anne 71, 102, 109

Niemann, B. 26

Niessen, M. 97

Nualart, F. 70

Numans, S. T. 91, 106

Ori, A. 89

Padvitski, T. 90

Pai, G. M. 69

Paudel, G. 86

Pendzialek, S. M. 102

Pfirrmann, Th. 85

Pierik, V. D. 91, 106

Pijnappels, M. 95, 97, 105, 107

Piskovatska, V. 92

Puls, A. 100

Quandt, D. 103

Reijnierse, E. M. 93, 94, 95, 96, 97

Rheinsberg, Z. 40

Rojer, A. G. M. 96, 97

Romani, N. 66

Rosenstock, Ph. 98

Rudolph, K. L. 42, 77

Ruhs, S. 78

Rujescu, D. 68, 74, 79, 100

Salazar, K. 70

Scheer, M. 72

Scheerman, K. 91, 106, 107

Scheffler, J. 99

Schiller, J. 79, 100

Schindler, M. 71, 102

Schmidt, R. 86

Schuetz, D. 101

Schönherr, R. 82

Sebekova, K. 24

Seeling, T. 71, 102

Seliger, B. 103

Shilyaev, N. 86

Simm, A. 12, 75, 80, 83, 88, 92, 99, 104, 110, 112, 113

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Sinz, A. 86

Sipilä, S. 95

Smolikova, G. 86

Somoza, V. 110

Spannbrucker, T. 59

Steinbrenner, H. 81

Stenroth, L. 95

Tarakhovskaya, E. 86

Tissier, A. 86

Toma, G. 103

Trappenburg, C. 93, 94, 95, 96, 97

Treede, H. 75, 83, 110

Tressel, E. 105

Unfried, K. 59

Urazova, A. 104

Urban, N. 81

van Ancum, J. M. 91, 105, 106, 107

van Lummel, R. C. 95, 97

van Rijssen, N. M. 96

van Schoor, N. M. 105

van Schooten, K. S. 97

Ventura, N. 108

Verlaan, S. 91, 93, 106, 107

Villavicencio-Lorini, P. 85

Vogt, Th. 86

Volk, Ch. 109

von Ameln, F. 60

Walentek, P. 85

Wedel, S. 66, 111

Wessjohann, L. A. 86

Wienke, A. 75

Winterhalter, P. 80, 104, 112

Wächter, K. 104, 110

Ye, Z. 113

Zurek, M. 60

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Thank’s to our Sponsors (in alphabetical order)

Bayer HealthCare 1,500 €

Biotronik Vertriebs GmbH & Co. KG 5,000 € Bristol-Myers Squibb 3.000 €

DFG up to 18,000 € Fumedica Medizintechnik GmbH 1,000 €

JR-herzchirurgische OP-Sets OHG 4,000 €

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Map of Halle (Saale)

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