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Screening of multiple gene mutations is also applicable for complex nervous and immune diseases
An extended application of the CRISPR-Cas technology has been made possible by Dr Manual Kaulich's team at Goethe University: the new 3Cs multiplex technique allows the effect of genetic changes in any two genes to be studied simultaneously in cell cultures. This can provide important clues for the development of therapies to treat cancer or diseases of the nervous and immune systems.
FRANKFURT. Cancer and many other diseases are based on genetic defects. The body can often compensate for the defect of one gene; it is only the combination of several genetic errors that leads to the clinical picture. The 3Cs multiplex technique based on CRISPR-Cas technology developed at Goethe University Frankfurt now offers a way to simulate millions of such combinations of genetic defects and study their effects in cell culture. These "gene scissors" make it possible to introduce, remove and switch off genes in a targeted manner. For this purpose, small snippets of genetic material ("single guide RNA") are used as "addresses" that guide the gene scissors to specific sections of the DNA, where the gene scissors then become active.
The scientists from the Institute of Biochemistry II at Goethe University have expanded the 3Cs technique that they developed and patented three years ago. 3Cs stands for covalently-closed circular-synthesised, because the RNA elements used for CRISPR-Cas are generated with the help of a circular synthesis and are thus distributed more uniformly. With a whole library of such RNA rings, any gene in a cell can be specifically addressed in order to change it or switch it off.
The new 3Cs multiplex technique now even allows the simultaneous manipulation of two genes in one cell. Dr. Manuel Kaulich explains: "We can produce 'address' RNA libraries for all conceivable two-gene combinations. This allows up to several million combinations to be tested simultaneously in one experiment."
Until now, the cost and effort of such experiments was very high; the research group's new technique reduces it, including costs, by a factor of ten. This is because the team can produce the address libraries very uniformly and in high quality thanks to the new 3Cs multiplex technique. "Due to the mediocre quality of the CRISPR-Cas libraries previously available, very large experiments always had to be carried out to statistically compensate for any errors that arose," says Kaulich.
Using the example of various genes involved in degradation processes, the research group demonstrated the potential of the new 3Cs multiplex technique: they examined almost 13,000 two-way combinations of genes that are responsible for recycling processes (autophagy) in the cell. With their help, the cell breaks down and recycles "worn-out" cell components. Disturbances in autophagy can trigger cell proliferation.
"Using the 3Cs multiplex technique, we were able to identify, for example, two genes involved in autophagy whose switching off leads to an uncontrolled growth of cells," explains Kaulich. "These are precisely the autophagy mutations that occur in every fifth patient with squamous cell carcinoma of the lung. In this way, we can search very efficiently in cell culture experiments for genes that play an important role in cancer, and also in diseases of the nervous and immune systems, and that are suitable as possible targets for therapies."
The Goethe University research group has applied for a patent for its developments through the university's technology transfer subsidiary Innovectis. The start-up company Vivlion GmbH, spun off from the Institute of Biochemistry II with the participation of Manuel Kaulich, is already offering the use of this technology on the market.
Publication: Valentina Diehl, Martin Wegner, Paolo Grumati, Koraljka Husnjak, Simone Schaubeck, Andrea Gubas, Varun Jayeshkumar Shah, Ibrahim H Polat, Felix Langschied, Cristian Prieto-Garcia, Konstantin Müller, Alkmini Kalousi, Ingo Ebersberger, Christian H Brandts, Ivan Dikic, Manuel Kaulich, Minimized combinatorial CRISPR screens identify genetic interactions in autophagy. Nucleic Acids Research, gkab309, https://doi.org/10.1093/nar/gkab309
Dr Manuel Kaulich
Institute for Biochemistry II
Goethe University Frankfurt
Tel: +49 69 6301-6295
Dr Kerstin Koch
Institute for Biochemistry II
Goethe University Frankfurt
Tel.: +49 696301-84250
Editor: Dr. Markus Bernards, Science Editor, PR & Communication Department, Tel: -49 (0) 69 798-12498,
Fax: +49 (0) 69 798-763 12531, email@example.com
German Research Foundation funds new CRC Transregio 326 „Geometry and arithmetic of uniformized structures” – CRC 1039 on medical signal path research enters third funding period – Goethe University involved in two further CRC-Transregios
Researching intricate geometric and arithmetic objects is the goal of the new Collaborative Research Centre Transregio 326 (TRR 326), coordinated by Goethe University. On 25th May, the German Research Foundation (DFG) announced that the TRR 326 would be funded with 9.2 million euros for the next four years. The CRC 1039 “Signalling by fatty acid derivatives and sphingolipids in health and disease", for which Goethe University is spokesperson, will be continued and receive 9.6 million euros for the third funding period. Two other TRRs in which Goethe University is involved will also be funded by the DFG: In the TRR 211 “Strongly interacting matter under extreme conditions", the spokesperson will switch from Goethe University to the Technical University of Darmstadt (9.2 million euros). Finally, scientists from Goethe University are also significantly involved in TRR 301 "The tropopause region in a changing atmosphere" (spokesperson: Johannes Gutenberg University Mainz, €12.3 million).
FRANKFURT. Professor Enrico Schleiff, President of Goethe University Frankfurt, congratulates the scientists on their success: "Goethe University's commitment, particularly in the Transregio Collaborative Research Centres, demonstrates our excellent scientific networking in the region, especially in the natural sciences and medicine. The association of the Rhine-Main universities of Frankfurt, Mainz and Darmstadt, have given this regional cooperation a framework: There are now more than 30 research associations and networks in this strategic alliance, and last year we established the RMU degree programme so that talented students can also benefit from RMU."
The mathematical exploration of complicated geometric and arithmetic spaces with the help of uniformization is the research topic of TRR 326 "Geometry and arithmetic of uniformized structures - GAUS". Together with coordinator Goethe University Frankfurt, the Technical University of Darmstadt and Heidelberg University successfully applied for TRR 326; associated institutions are the Johannes Gutenberg University Mainz and the Technical University of Munich. The concept of uniformization goes back to ideas of Felix Klein and Henri Poincaré from the 19th century and seeks a uniform description of certain geometric objects. A very simple example uniformization can be illustrated with the slinky, a metal spiral toy which is able to "run" down a staircase doing "somersaults". When pressed together, it has - seen from above - the geometry of a circle. This circle can be uniformized by pulling the metal spiral apart. It becomes particularly simple when the spiral is completely unwound and, geometrically, is only a simple wire. In order to preserve the information of the slinky, each spiral turn on the wire is marked with a colour dot, which gives the wire a shifting symmetry (you change levels in the spiral). A globally complicated geometric space (in our example, the circle of the slinky) is replaced by a much simpler space (here a straight line) without changing the local structure. The original complexity is described by internal symmetries (illustrated in the example by periodic markings) of the simpler space.
In TRR 326 GAUS, mathematicians deal with uniformization of very complicated geometric spaces - this includes modern geometric concepts, in particular tropical and p-adic geometries - and with analogous applications of the uniformization technique to arithmetic (number-theoretic) questions. Here, the researchers try to identify fundamental connections, for example to moduli spaces, automorphic forms, Galois representations, and cohomological structures. Professor Jakob Stix, mathematician at Goethe University and GAUS spokesman, says: "With the SFB Transregio GAUS, we are building on the extremely successful collaboration between TU Darmstadt and Goethe University in the LOEWE priority 'Uniformed Structures in Arithmetic and Geometry' and the DFG research group 'Symmetry, Geometry and Arithmetic' at TU Darmstadt and Heidelberg University. I am very much looking forward to doing joint research with so many outstanding colleagues."
The Collaborative Research Centre 1039 "Signalling by fatty acid derivatives and sphingolipids in health and disease," which Goethe University is now continuing together with the Max Planck Institute for Heart and Lung Research in Bad Nauheim, is entering its third funding period. The scientists are studying a group of poorly water-soluble biomolecules, the lipids. As lipid bilayers, they prominently form the membranes that surround our cells and also divide the interior of the cells. As fats, they serve as energy storage for our bodies.
However, CRC 1039 is investigating a function that is still comparatively under-researched: Lipids are part of many signalling pathways through which cells regulate growth and metabolism and communicate with their environment. Dysregulated lipids are apparently decisively involved in the development and progression of diseases such as diabetes, cancer, inflammation, and neurodegenerative diseases. After fundamental work in the first two funding periods, the third funding period focuses on understanding the whole organism. Professor Josef Pfeilschifter, pharmacologist at Goethe University and spokesman of SFB 1039, explains: "We want to understand the lipid signalling network as a whole and thus develop innovative ways to diagnose and treat a wide variety of diseases related to dysregulated lipids. In doing so, we can rely on a long-standing and broad expertise in 'lipid signalling', which is also founded on the establishment of sophisticated analytical methods based on mass spectrometry."
Scientists from Goethe University are significantly involved in two other CRC Transregios:
How matter behaves under conditions of extreme pressure and temperature, in which atoms overlap and fuse with each other is being investigated by TRR 211 "Strongly interacting matter under extreme conditions", which is entering its second funding period. For extremely short periods of time, such states of matter can be created in particle accelerators, revealing something about the strong interaction that holds atomic nuclei together. In the cosmos, such extreme states of matter occur when, for example, neutron stars collide with each other. BesidesGoethe University, the Technical University of Darmstadt, which is the new host university, and Bielefeld University are also involved in this collaborative research centre.
In the new TRR 301 "The tropopause region in a changing atmosphere", atmospheric scientists will study the tropopause region: the zone in the atmosphere that separates the lower "weather layer" (troposphere) from the stratosphere above. The research focus is on the physical and chemical processes of this region and their influence on planetary circulation and climate. The main locations are the Johannes Gutenberg University Mainz (spokesperson) and Goethe University Frankfurt. Also involved are the Technical University of Darmstadt, the Ludwig Maximilian University of Munich, the Max Planck Institute for Chemistry in Mainz, the Jülich Research Centre and the German Aerospace Centre (DLR) in Oberpfaffenhofen.
Image for download:
Caption: Using the mathematical technique of uniformization, complicated geometric spaces (here: the j-invariant as an automorphic function on the uniformization of the moduli space of elliptic curves) can be represented as highly symmetric geometric patterns. Credit: Michaelis Neururer
Professor Josef Pfeilschifter
Spokesman SFB 1039 “Signalling by fatty acid derivatives and sphingolipids in health and disease"
Institute for General Pharmacology and Toxicology
Goethe University Frankfurt
Tel. +49 69 6301-6950
TRR 211 „Strongly
interacting matter under extreme conditions“
301 „The tropopause region in a changing atmosphere“
Fear of long-term consequences – physical activity could actually help to better manage the pandemic
During the first lockdown people were a good 40 percent less active, as shown by an international study led by Goethe University Frankfurt. Psychological well-being sank as well; the portion of people at potential risk for depression tripled. The authors fear long-term consequences and urge that this be taken into account going forward.
FRANKFURT. Twenty scientists from 14 countries warn of a hidden “pandemic within the pandemic“ in two current publications. On the one hand, physical activity levels have gone down significantly, on the other hand, psychological well-being has suffered. “Governments and those responsible for health systems should take our findings seriously," emphasizes the author team, headed by Dr Jan Wilke from the Institute for Sport Sciences at Goethe University Frankfurt.
About 15,000 people in participating countries answered standardised questionaires as part of an international survey. In April/May 2020, they reported physical activity levels (13,500 participants) as well as their mental and physical well-being (15,000 participants) before and during the pandemic-related restrictions.
Older individuals especially affected
“The results show drastic reductions in physical activity and well-being," says Wilke. More than two thirds of those questioned were unable to maintain their usual level of activity. Moderate exercise decreased by an average of 41 percent according to self-reported data - this includes anything that increases heart rate and breathing, such as brisk walking, running, cycling or even strenuous gardening.
The proportion of vigorous exercise during which people sweat and clearly run out of breath fell by a similar amount (42 percent). The effects were somewhat higher among professional athletes and particularly active people, as well as comparatively young and old people. The decline in activity was particularly noticeable among people over 70 years of age, who were 56 to 67 percent less active than before. "We know that physical inactivity, especially in older people, can lead to changes that are difficult to reverse after only two weeks - for example, in body fat percentage or insulin sensitivity," warn the study authors.
Exercise helps prevents disease and reduces mortality
The WHO recommends at least 150 minutes of moderate or 75 minutes of intensive physical activity per week - 81 percent of the study participants achieved this before the pandemic, but only 63 percent during the lockdowns. Yet sufficient exercise can reduce mortality by up to 39 per cent, as a 2015 study showed. Data suggests that too little exercise plays a role in about one in ten premature deaths, because physical activity reduces the likelihood of, for example, high blood pressure, metabolic disorders such as type 2 diabetes, and cancer.
Exercise is known to activate the immune system because it promotes blood circulation and activates lymphocytes and messenger substances (cytokines) that are important for immune defence. Studies show that physically active people are less susceptible to influenza, rhino and herpes viruses and respiratory infections in general. So it may be that exercise also offers protection against severe COVID-19 by reducing risk factors such as obesity. Physical health and exercise also reduce the risk of mental health problems such as depression and anxiety disorders.
Mental well-being drastically reduced
In another part of the study, the team of authors asked about mental well-being during the pandemic restrictions. 73 percent of the study participants stated that their well-being had deteriorated. The perceived quality of life as measured by the WHO well-being Index, which measures mood, relaxation, activity, rest and interest, dropped on average from 68 percent before the pandemic to 52 percent during the first lockdown phase.
Above all, people felt less "active and full of energy" and led a life less "filled with interesting things". The proportion of very low scores indicating a possible risk of depression tripled from 15 to 45 percent. "These effects were stronger among women and younger people, " the study says. "More attention should be paid to the needs of women in particular, as they are significantly more vulnerable."
Nonetheless, 14 to 20 percent of the respondents also stated that their health had improved - the authors see more family time, greater work autonomy, fewer business trips or a changed perception of health as possible reasons. "But a large part of the population may still be suffering from barely visible health effects of the pandemic," the team of authors warns.
This could also translate into rising health costs: According to US data, the annual expenditure for inactive or insufficiently active people increases by 1200 and 600 euros respectively - this would add up to two to four million euros after one year just for the 3104 people from the survey who did not exercise enough during the lockdown.
The results of these first multinational studies are likely to be relevant for an estimated four billion people worldwide who were affected by the restrictions of the first coronavirus wave in the spring of 2020. However, the data was predominantly collected through electronic media, so populations without internet were not included. Also, no differentiation was made according to factors such as living environment, education and social status. In addition, the data is based on self-assessments, not measurements, which may distort retrospective perceptions in particular. "Nevertheless, our results show that the issues of physical activity and well-being belong on the policy agenda," Wilke emphasises.
"Governmental and health-related decison-makers need to develop strategies to mitigate the loss of physical activity," write the authors. They suggest better public education, creating exercise opportunities with a low likelihood of infection, or offering effective home exercise programmes. Among numerous other health facets, this would have a particularly positive effect on mental well-being.
Negative effects similar to those observed in these studies should be avoided at all costs in future pandemics. "Unfortunately, physical activity and exercise do not have a strong lobby and are usually neglected in public discourse," says Wilke. "Yet they can greatly help us to better cope with the pandemic."
Publications: Jan Wilke et al. A Pandemic within the Pandemic? Physical Activity Levels Substantially Decreased in Countries Affected by COVID-19. Int. J. Environ. Res. Public Health, Vol. 18, 5 (2021), https://www.mdpi.com/1660-4601/18/5/2235/htm#B11-ijerph-18-02235
Jan Wilke et al., Drastic Reductions in Mental Well-Being Observed Globally During the COVID-19 Pandemic: Results from the ASAP Survey. Front. Med. 8:578959 (2021), https://www.frontiersin.org/articles/10.3389/fmed.2021.578959/full
Dr Jan Wilke
Institute für Sport Sciences
Goethe University Frankfurt
Tel. +49 (69) 798-24588,
Editor: Dr Markus Bernards, Science Editor, PR & Communication Department, Tel: +49 (0) 69 798-12498, Fax: +49 (0) 69 798-763 12531, firstname.lastname@example.org
Scientists at Goethe University Frankfurt and the Event Horizon Telescope Collaboration use data which produced the first image of a black hole to constrain its fundamental properties.
Theoretical physicists at Goethe University Frankfurt have analysed data from the black hole M87* as part of the Event Horizon Telescope (EHT) collaboration to test Albert Einstein's theory of general relativity. According to the tests, the size of the shadow from M87* is in excellent agreement being from a black hole in general relativity, but sets constraints on the properties of black holes in other theories. In 2019, the EHT collaboration published the first image of a black hole located at the centre of the galaxy M87.
FRANKFURT. As first pointed out by the German astronomer Karl Schwarzschild, black holes bend space-time to an extreme degree due to their extraordinary concentration of mass, and heat up the matter in their vicinity so that it begins to glow. New Zealand physicist Roy Kerr showed rotation can change the black hole's size and the geometry of its surroundings. The "edge" of a black hole is known as the event horizon, the boundary around the concentration of mass beyond which light and matter cannot escape and which makes the black hole “black". Black holes, theory predicts, can be described by a handful of properties: mass, spin, and a variety of possible charges.
In addition to black holes predicted from Einstein's theory of general relativity, one can consider those from models inspired by string theories, which describe matter and all particles as modes of tiny vibrating strings. String-inspired theories of black holes predict the existence of an additional field in the description of fundamental physics, which leads to observable modifications in the sizes of black holes as well as in the curvature in their vicinity.
Physicists Dr Prashant Kocherlakota and Professor Luciano Rezzolla from the Institute for Theoretical Physics at Goethe University Frankfurt, have now investigated for the first time how the different theories fit with the observational data of the black hole M87* at the centre of the galaxy Messier 87. The image of M87*, taken in 2019 by the international Event Horizon Telescope (EHT) collaboration, was the first experimental proof of the actual existence of black holes after the measurement of gravitational waves in 2015.
The result of these investigations: The data from M87* are in excellent agreement with the Einstein-based theories and to a certain extent with the string-based theories. Dr Prashant Kocherlakota explains: "With the data recorded by the EHT collaboration, we can now test different theories of physics with black hole images. Currently, we cannot reject these theories when describing the shadow size of M87*, but our calculations constrain the range of validity of these black hole models."
Professor Luciano Rezzolla says: “The idea of black holes for us theoretical physicists is at the same time a source of concern and of inspiration. While we still struggle with some of the consequences of black holes – such as the event horizon or the singularity – we seem always keen to find new black hole solutions also in other theories. It is therefore very important to obtain results like ours, which determine what is plausible and what is not. This was an important first step and our constraints will be improved as new observations are made".
In the Event Horizon Telescope collaboration, telescopes from around the globe are interconnected to form a virtual giant telescope with a dish as big as the Earth itself. With the precision of this telescope, a newspaper in New York could be read from a street café in Berlin.
Publication: Prashant Kocherlakota, Luciano Rezzolla, Heino Falcke, Christian M. Fromm, Michael Kramer, Yosuke Mizuno, Antonios Nathanail, H´ector Olivares, Ziri Younsi et. al. (The Event Horizon Telescope collaboration), Constraints on black-hole charges with the 2017 EHT observations of M87*. Physical Review D, vol 103, https://journals.aps.org/prd/abstract/10.1103/PhysRevD.103.104047 DOI: 10.1103/PhysRevD.103.104047
Testing different theories of gravity with
the data obtained
Caption: Event horizon sizes for different theories of gravity. All of these black holes cast dark shadows that are distinguishable from each other in size, but only those that fall in the gray band are compatible with the 2017 EHT measurements of M87*, and in this image, the one represented in red at the bottom is too small to be a viable model for M87*. Credit: Prashant Kocherlakota, Luciano Rezzolla (Goethe University Frankfurt and EHT Collaboration/ Fiks Film 2021)
Dr Prashant Kocherlakota
Institute for Theoretical Physics
Goethe University Frankfurt
Tel. +49 69 798-47848
Professor Luciano Rezzolla
Institute for Theoretical Physics
Goethe University Frankfurt
Editor: Dr. Markus Bernards, Science Editor, PR & Communication Department, Tel: -49 (0) 69 798-12498, Fax: +49 (0) 69 798-763 12531, E-Mail: email@example.com
How novel therapeutics provide insight into bacteria membranes
Whether bacteria are resistant to antibiotics is often decided at the cell membrane. This is where antibiotics can be blocked on their way into the cell interior or catapulted from the inside to the outside. Macrocyclic peptides, a novel class of antibiotics, bioactive cytotoxins and inhibitors, shed light on how this transport process occurs at the membrane, how it is influenced and how it can be used to circumvent the resistance of a malignantly transformed cell. The research results, which were developed under the direction of Professor Robert Tampé (Goethe University) and Professor Hiroaki Suga (University of Tokyo), have been published in the renowned journal eLife (20-02-2021-RA-eLife-67732).
FRANKFURT. There are currently only a few synthetic agents that bind to and block the widespread membrane transport proteins, ATP-binding cassette transporters (ABC). Scientists at Goethe University and the University of Tokyo identified four of these macrocyclic peptides as models for a novel generation of active substances. They used methods for which the scientists involved are considered world leaders.
Thanks to deep sequencing, an extremely fast and efficient read-out procedure, the desired macrocyclic peptides could be filtered out of a "library" of macrocyclic peptides comprising trillions of variants (1 with 12 zeroes) - a number that exceeds the number of stars in the Milky Way. The fact that such an enormous amount exists at all is related to a novel procedure: By reprogramming the genetic code, amino acids can be used specifically as active components that are not otherwise used in the cell. In particular, their circular, closed structure distinguishes them from natural proteins. "Because these therapeutics are cyclic, they break down less rapidly in the cell," explains Robert Tampé, Director of the Institute of Biochemistry at Goethe University. "In addition, the ring-shaped active substances are restricted in their spatial structure, so they bind to the target molecule without major rearrangements." A third distinguishing feature makes macrocyclic peptides particularly attractive for scientists: When the active substances are produced, their building instructions are supplied as a "barcode". If certain therapeutics are selected from among a trillion synthetically produced ones, they carry their "name tags" with them, so to speak.
So what role do synthetic therapeutics play in antibiotic resistance in bacteria or multidrug resistance in tumour cells? What happens when they encounter the ATP-driven transport molecule that is responsible for resistance by carrying the chemotherapeutic agents out of the cell? In a nutshell: The drugs block the transporter by binding to it. This can happen at the beginning or at the end of a transport process, when the transporter is in a resting state. However, since the scientists can slow down the transport process so that it is carried out in slow motion, they can identify the agents that "enter" in the middle of the transport process and "hold" the membrane protein in its respective position. In this way, the researchers gain an insight into the choreography of the transport process as if through the images of a film strip.
These insights have already led to a "paradigm shift" in science, as Tampé explains: "Until now, we have assumed that ATP hydrolysis (note: an energy-releasing splitting process) provides the energy for transport through the membrane. However, this is only indirectly the case. It is the event of the binding of the ATP molecule that pushes substances out of the cell. The energy of hydrolysis, on the other hand, is used to return the ABC transporter to its initial state." The research groups at Goethe University and the University of Tokyo are convinced that these and other insights into membrane processes will point to the development of future medicines.
Basic research on cellular membranes and membrane proteins already has a long tradition in Frankfurt. Robert Tampé elucidated essential mechanisms of ATP-driven transport proteins and cellular machinery of adaptive immune response and quality control, which together with this new publication can provide approaches for applied drug research. Tampé was head of the Collaborative Research Centre "Transport and Communication across Biological Membranes" (SFB 807) which expired at the end of 2020. Meanwhile the concept for a new research centre on highly dynamic processes related to protein networks and machineries in cellular membranes is already under development. In the long term, the research results should reveal new possibilities for the therapy of molecular diseases, infections and cancer.
Erich Stefan, Richard Obexer, Susanne Hofmann, Khanh Vu Huu, Yichao Huang, Nina Morgner, Hiroaki Suga, Robert Tampé: “De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC transporter by multimode allosteric inhibition“ (20-02-2021-RA-eLife-67732)
Stefan, Hofmann, and Tampé at the Institute of Biochemistry at Goethe University, Vu Huu and Morgner at the Institute for Physical and Theoretical Chemistry at Goethe University, and Obexer, Huang and Suga at the Department of Chemistry, University of Tokyo.
Images for download:
(Graphic: Robert Tampé, Institute for Biochemistry, Biocentre, Goethe University Frankfurt)
Caption: Synthetic therapeutics for antibiotic resistance in bacteria or multidrug resistance in tumour cells can block ATP-driven transport proteins that carries chemotherapeutics out of the cell
Professor Robert Tampé
Institute of Biochemistry, Biocentre
Goethe University Frankfurt
Department of Chemistry
Graduate School of Science
The University of Tokyo
Editor: Pia Barth, Public Relations, PR & Communication Department, Tel: -49 (0) 69 798-12481, Fax: +49 (0) 69 798-763 12531, firstname.lastname@example.org