Poliovirus und SARS-CoV-2: Mechanochemie und Inhibitoren aus der Einzelmolekülperspektive

Viruses, especially emerging and re-emerging positive-sense single-stranded RNA ((+)ssRNA) viruses, such as the severe acute respiratory syndrome coronavirus - 2 (SARS-CoV-2), have caused several epidemic and pandemic outbreaks in the last decades. There are only few approved antiviral drugs available against (+)ssRNA viruses and they rarely show sufficient efficacy when repurposed against emerging viruses. The major drug target in viruses is the viral replication machinery, i.e. the replication-transcription complex (RTC), which is responsible for the synthesis of the viral genome and as such is a vital part in the replication cycle of the virus. Most antiviral drugs that target the RTC are nucleotide analogs, which are modified natural nucleotides that can be used as substrate for RNA synthesis by the RTC. They can e.g. inhibit RNA product extension or induce mutations. To find (new) effective nucleotide analogs against the viral RTC, it is necessary to understand how the RTC incorporates naturally occurring nucleotides, which parts of this process are influenced by the incorporation of nucleotide analogs and how the nucleotide analogs affect RNA synthesis dynamics. The incorporation of a nucleotide analog is a rare event, similar to mismatch incorporation, that happens asynchronous between different RTCs. These events are hidden in standard biochemical bulk assays in the presence of competing natural nucleotides at physiological concentration. Single-molecule techniques by contrast allow for the observation of single enzymes, which in turn enables us to observe rare events, even in the presence of saturating concentrations of the competing natural nucleotides. Here, we used high-throughput magnetic tweezers as a single molecule force spectroscopy technique for the simultaneous observation of hundreds of RNA template, which therefore allows for the rapid collection of large statistics. A pair of permanent magnets exerts an attractive force to magnetic beads, which are tethered to the surface of a flow chamber by the template RNAs. The RTC’s RNA elongation activity modifies the end-to-end extension of the tether, and therefore the vertical position of the magnetic bead, which we tracked in real-time using a CMOS camera. We used the position of the bead to extract the RTC kinetics based on first-passage analysis and Maximum Likelihood Estimation. To generate a large number of available templates per experiment, we optimized the fabrication method to produce hairpin and linear RNA constructs that are suitable template for RTC assembly and elongation and provide a visible change in extension during the experiment with high purity and yield. As the temperature, at which viral RTCs naturally work, is often above room temperature, we outfitted our magnetic tweezers assay with a heating device and calibrated the temperature precisely using DNA as a temperature sensor. As proof of principle, we used our magnetic tweezers assay to investigate the influence of temperature on the RNA synthesis activity of poliovirus RNA-dependent RNA polymerase (RdRp), for which the mechanochemical mechanism has already been investigated in depth. We then used our temperature-controlled high-throughput magnetic tweezers assay to study SARS-CoV-2 replication. Based on the effects of force and nucleotide concentration on the RNA synthesis dynamics, we were able to develop a kinetic model for nucleotide addition, containing three catalytic pathways. These pathways only differ in the duration, while the steps in the nucleotide addition cycles is conserved for the three pathways. We also provide the first evidence that the translocation step, i.e. the downstream movement of the SARS-CoV-2 polymerase on the template, is based on a thermal ratchet mechanism and not a power stroke. Based on these results, we investigated the effect of several nucleotide analogs, which have been suggested as drugs against SARS-CoV-2 infections. Our single molecule investigation revealed that 2′ and 3′ ribose modified nucleotide analogues are incorporated via the main fast catalytic pathways, while base- and 1′ ribose modified nucleotide analogues were incorporated via the two slow catalytic pathways. Furthermore, we demonstrated that Remdesivir, the only FDA approved drugs to treat SARS-CoV-2 infection does not terminate RNA synthesis, but rather induces long pauses related to polymerase backtracking. To summarize, this thesis describes the development of a temperature-controlled high-throughput magnetic tweezers assay and high quality RNA template to elucidate the nucleotide addition cycle of SARS-CoV-2 and the identification of the mechanism of action and efficacy of several nucleotide analogs. The results pave the way towards high spatiotemporal characterization and therefore an in-depth fundamental understanding the mechanism of action of antiviral drugs.Viren, insbesondere neu- oder wiederauftretenden (+)ssRNA Viren, wie das severe acute respiratory syndrome coronavirus-2, haben in den vergangenen Jahrzehnten viele Enepidemien und -pandemien verursacht. Es gibt nur wenige zugelassene antivirale Medikamente gegen (+)ssRNA Viren und diese zeigen selten ausreichende Wirksamkeit, wenn sie gegen neuauftretende Viren verwendet werden. Als Angriffspunkt für antivirale Medikamente eignet sich besonders die virale Replikationsmaschinerie (eng. replication-transcription complex, RTC), die für die Synthese des gesamten viralen Genoms verantwortlich und somit ein zentraler Bestandteil des viralen Replikationszyklus ist. Die meisten Wirkstoffe, die den RTC angreifen, sind sogenannte Nukleotidanaloga, natürlich vorkommende Nukleotide, die modifiziert wurden und von dem RTC als Substrat für die RNA Synthese verwendet werden können. Um (neue) wirksame Nukleotidanaloga gegen einen bestimmten Virus zu finden, muss man zunächst verstehen, wie der RTC natürlich vorkommende Nukleotide einbaut, welche Abschnitte dieses Prozesses durch Nukleotidanaloga beeinflusst werden und wie Nukleotidanaloga die RNA-Synthesedynamik beeinflusst. Der Einbau eines Nukleotidanalogons durch den RTC stellt ein seltenes Ereignis dar, ähnlich dem Einbau einer Nukleotidfehlpaarung, das bei den RTCs asynchron stattfindet. Diese seltenen Ereignisse sind bei biochemischen Standardmessungen in Messungen mit konkurrierenden natürlich vorkommenden Nukleotiden nicht sichtbar. Bei Einzelmolekülmethoden hingegen können einzelne Enzyme beobachtet werden, was es wiederum erlaubt auch unter Bedingungen mit saturierenden Konzentrationen an natürlich vorkommenden Nukleotiden seltene Ereignisse zu beobachten. Hier haben wir eine temperaturkontrollierte, hochdurchsätzende magnetische Pinzette als Einzelmolekül-Kraftspektroskopie Methode verwendet, um hunderte RTCs in parallel zu beobachten, was es uns ermöglichte schnell eine große Menge an Stichproben zu messen. Ein Paar permanenter Magneten übt eine anziehende Kraft auf magnetische Kügelchen aus, die durch Matrizen-RNAs mit der Oberfläche einer Durchflusskammer verbunden sind. Durch die RNA-Syntheseaktivität des RTC ändert sich die Ende-zu-Ende Distanz der RNA und dadurch bedingt die vertikale Position des magnetischen Kügelchens, die wir in Echtzeit mit einer CMOS Kamera verfolgten. Wir verwendeten diese Position um die Kinetik des RTC mithilfe der Erstpassagezeitanalyse und der Größte-Dichte-Methode. Um eine möglichst große Anzahl an Matrizensträngen pro Experiment zur Verfügung zu haben, haben wir die herstellungsmethoden optimiert, um lineare RNA und RNA-Haarnadelstrukturen n großer Anzahl und Reinheit, die geeignete Matrizen für den Zusammenbau des RTC und dessen Elongationsaktivität darstellen und eine sichtbare Änderung ihrer Länge während des Experiments zeigen, zu produzieren. Da die Temperaturen, bei denen der virale RTC normalerweise arbeitet, oft über der Raumtemperatur sind, haben wir unsere magnetische Pinzette mit einer Heizung ausgestattet und diese präzise mithilfe einer DNA-Sonde kalibriert Als Machbarkeitsnachweis haben wir unsere magnetische Pinzette genutzt, um den Einfluss von Temperatur auf das RNA–Syntheseverhalten der Replikationsmaschinerie des Poliovirus, der RNA-abhängigen RNA Polymerase (eng. RNA-dependent RNA polymerase, RdRp), zu untersuchen, für die die mechanochemische Funktionsweise bereits im Detail untersucht wurde. Im Anschluss verwendeten wir die temperaturkontrollierte, hochdurchsätzende magnetische Pinzette, um die SARS-CoV-2 Replikation zu untersuchen. Ausgehend von dem beobachteten Einfluss von Kraft und Nukleotidkonzentration auf die RNA-Syntheseaktivität haben wir ein Modell für die Nukleotidaddition erstellt, das drei katalytisch aktive Pfade beinhaltet. Diese Pfade variieren nur in ihrer Dauer, während die einzelnen Schritte gleich sind. Wir lieferten außerdem den ersten Beweis, dass die Translokation, also die strangabwärtige Bewegung der SARS-CoV-2 Polymerase, auf einer molekularen Ratsche und nicht auf einem Kraftschlag basiert. Ausgehend von unseren Resultaten untersuchten wir den Effekt verschiedener Nukleotidanaloga auf die SARS-CoV-2 Polymerase, die als Medikamente gegen SARS-CoV-2 Infektionen vorgeschlagen wurden. Unsere Einzelmolekülstudie zeigte, dass Nukleotidanaloga mit Modifikationen an 2′ oder 3′ Positionen der Ribosedurch den schnellen Hauptpfad eingebaut werden, während Nukleotidanaloga mit Modifikationen an der 1′ Position der Ribose oder an der Base über die beiden langsameren Pfade eingebaut werden. Wir konnten außerdem zeigen, dass Remdesivir, das einzige momentan von der FDA zugelassene Medikament gegen SARS-COV-2 Infektionen, nicht wie bislang angenommen zu einem verspäteten Kettenabbruch führt sondern langlebige Synthesepausen mit Rückwärtsbewegung (backtrack) der SARS-CoV-2 Polymerase auf der RNA verursacht. Zusammengefasst wird in dieser Arbeit die Entwicklung einer temperaturkontrollierten, hochdurchsätzenden magnetischen Pinzette und hochwertiger RNA Konstrukte beschrieben, mithilfe derer der Nukleotidadditionszyklus der SARS-CoV-2 Polymerase aufgeklärt wurde und sowohl Wirkmechanismus als auch Wirksamkeit verschiedener Nukleotidanaloga bestimmt wurde. Diese Resultate ebnen den Weg für eine hochaufgelöste räumlich-zeitliche Charakterisierung, und damit auch ein grundlegendes Verstäntnis, des Wirkmechanismus antiviraler Medikamente

Temperature-dependent twist of double-stranded RNA probed by magnetic tweezer experiments and molecular dynamics simulations

RNA plays critical roles in the transmission and regulation of genetic information and is increasingly used in biomedical and biotechnological applications. Functional RNAs contain extended double-stranded regions, and the structure of double-stranded RNA (dsRNA) has been revealed at high resolution. However, the dependence of the properties of the RNA double helix on environmental effects, notably temperature, is still poorly understood. Here, we use single-molecule magnetic tweezer measurements to determine the dependence of the dsRNA twist on temperature. We find that dsRNA unwinds with increasing temperature, even more than DNA, with ΔTwRNA = −14.4 ± 0.7°/(°C·kbp), compared to ΔTwDNA = −11.0 ± 1.2°/(°C·kbp). All-atom molecular dynamics (MD) simulations using a range of nucleic acid force fields, ion parameters, and water models correctly predict that dsRNA unwinds with rising temperature but significantly underestimate the magnitude of the effect. These MD data, together with additional MD simulations involving DNA and DNA–RNA hybrid duplexes, reveal a linear correlation between the twist temperature decrease and the helical rise, in line with DNA but at variance with RNA experimental data. We speculate that this discrepancy might be caused by some unknown bias in the RNA force fields tested or by as yet undiscovered transient alternative structures in the RNA duplex. Our results provide a baseline to model more complex RNA assemblies and to test and develop new parametrizations for RNA simulations. They may also inspire physical models of the temperature-dependent dsRNA structure

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25 degrees C and 45 degrees C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Phi 6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Phi 6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.Peer reviewe

Temperature-Dependent Twist of Double-Stranded RNA Probed by Magnetic Tweezer Experiments and Molecular Dynamics Simulations

RNA plays critical roles in the transmission and regulation of genetic information and is increasingly used in biomedical and biotechnological applications. Functional RNAs contain extended double-stranded regions, and the structure of double-stranded RNA (dsRNA) has been revealed at high resolution. However, the dependence of the properties of the RNA double helix on environmental effects, notably temperature, is still poorly understood. Here, we use single-molecule magnetic tweezer measurements to determine the dependence of the dsRNA twist on temperature. We find that dsRNA unwinds with increasing temperature, even more than DNA, with ΔTwRNA = −14.4 ± 0.7°/(°C·kbp), compared to ΔTwDNA = −11.0 ± 1.2°/(°C·kbp). All-atom molecular dynamics (MD) simulations using a range of nucleic acid force fields, ion parameters, and water models correctly predict that dsRNA unwinds with rising temperature but significantly underestimate the magnitude of the effect. These MD data, together with additional MD simulations involving DNA and DNA-RNA hybrid duplexes, reveal a linear correlation between the twist temperature decrease and the helical rise, in line with DNA but at variance with RNA experimental data. We speculate that this discrepancy might be caused by some unknown bias in the RNA force fields tested or by as yet undiscovered transient alternative structures in the RNA duplex. Our results provide a baseline to model more complex RNA assemblies and to test and develop new parametrizations for RNA simulations. They may also inspire physical models of the temperature-dependent dsRNA structure

Four-dimensional distribution of the 2010 Eyjafjallajökull volcanic cloud over Europe observed by EARLINET

© Author(s) 2013. This work is distributed under the Creative Commons Attribution 3.0 License.The eruption of the Icelandic volcano Eyjafjallaj ökull in April-May 2010 represents a "natural experiment" to study the impact of volcanic emissions on a continental scale. For the first time, quantitative data about the presence, altitude, and layering of the volcanic cloud, in conjunction with optical information, are available for most parts of Europe derived from the observations by the European Aerosol Research Lidar NETwork (EARLINET). Based on multi-wavelength Raman lidar systems, EARLINET is the only instrument worldwide that is able to provide dense time series of high-quality optical data to be used for aerosol typing and for the retrieval of particle microphysical properties as a function of altitude. In this work we show the four-dimensional (4-D) distribution of the Eyjafjallajökull volcanic cloud in the troposphere over Europe as observed by EARLINET during the entire volcanic event (15 April-26 May 2010). All optical properties directly measured (backscatter, extinction, and particle linear depolarization ratio) are stored in the EARLINET database available at www.earlinet.org. A specific relational database providing the volcanic mask over Europe, realized ad hoc for this specific event, has been developed and is available on request at www.earlinet.org. During the first days after the eruption, volcanic particles were detected over Central Europe within a wide range of altitudes, from the upper troposphere down to the local planetary boundary layer (PBL). After 19 April 2010, volcanic particles were detected over southern and south-eastern Europe. During the first half of May (5-15 May), material emitted by the Eyjafjallajökull volcano was detected over Spain and Portugal and then over the Mediterranean and the Balkans. The last observations of the event were recorded until 25 May in Central Europe and in the Eastern Mediterranean area. The 4-D distribution of volcanic aerosol layering and optical properties on European scale reported here provides an unprecedented data set for evaluating satellite data and aerosol dispersion models for this kind of volcanic events.Peer reviewe

Tropospheric and stratospheric smoke over Europe as observed within EARLINET/ACTRIS in summer 2017

For several weeks in summer 2017, strong smoke layers were observed over Europe at numerous EARLINET stations. EARLINET is the European research lidar network and part of ACTRIS and comprises more than 30 ground-based lidars. The smoke layers were observed in the troposphere as well as in the stratosphere up to 25 km from Northern Scandinavia over whole western and central Europe to the Mediterranean regions. Backward trajectory analysis among other tools revealed that these smoke layers originated from strong wild fires in western Canada in combination with pyrocumulus convection. An extraordinary fire event in the mid of August caused intense smoke layers that were observed across Europe for several weeks starting on 18 August 2017. Maximum aerosol optical depths up to 1.0 at 532 nm were observed at Leipzig, Germany, on 22 August 2017 during the peak of this event. The stratospheric smoke layers reached extinction coefficient values of more than 600 Mm−1 at 532 nm, a factor of 10 higher than observed for volcanic ash after the Pinatubo eruption in the 1990s. First analyses of the intensive optical properties revealed low particle depolarization values at 532 nm for the tropospheric smoke (spherical particles) and rather high values (up to 20%) in the stratosphere. However, a strong wavelength dependence of the depolarization ratio was measured for the stratospheric smoke. This indicates irregularly shaped stratospheric smoke particles in the size range of the accumulation mode. This unique depolarization feature makes it possible to distinguish clearly smoke aerosol from cirrus clouds or other aerosol types by polarization lidar measurements. Particle extinction-to-backscatter ratios were rather low in the order of 40 to 50 sr at 355 nm, while values between 70-90 sr were measured at higher wavelengths. In the western and central Mediterranean, stratospheric smoke layers were most prominent in the end of August at heights between 16 and 20 km. In contrast, stratospheric smoke started to occur in the eastern Mediterranean (Cyprus and Israel) in the beginning of September between 18 and 23 km. Stratospheric smoke was still visible in the beginning of October at certain locations (e.g. Evora, Portugal), while tropospheric smoke was mainly observed until the end of August within Europe. An overview of the smoke layers measured at several EARLINET sites will be given. The temporal development of these layers as well as their geometrical and optical properties will be presented

Executive functions and borderline personality features in adolescents with major depressive disorder

BackgroundExecutive functions (EF) consolidate during adolescence and are impaired in various emerging psychiatric disorders, such as pediatric Major Depressive Disorder (pMDD) and Borderline Personality Disorder. Previous studies point to a marked heterogeneity of deficits in EF in pMDD. We examined the hypothesis that deficits in EF in adolescents with pMDD might be related to comorbid Borderline Personality features (BPF).MethodsWe examined a sample of 144 adolescents (15.86 ± 1.32) diagnosed with pMDD. Parents rated their child’s EF in everyday life with the Behavior Rating Inventory of Executive Function (BRIEF) and BPF with the Impulsivity and Emotion Dysregulation Scale (IED-27). The adolescents completed equivalent self-rating measures. Self- and parent-ratings of the BRIEF scores were compared with paired t-Tests. Correlation and parallel mediation analyses, ICC, and multiple regression analyses were used to assess symptom overlap, parent-child agreement, and the influence of depression severity.ResultsOver the whole sample, none of the self- or parent-rated BRIEF scales reached a mean score above T > 65, which would indicate clinically impaired functioning. Adolescents tended to report higher impairment in EF than their parents. Depression severity was the strongest predictor for BPF scores, with Emotional Control predicting parent-rated BPF and Inhibit predicting self-rated BPF. Furthermore, the Behavioral Regulation Index, which includes EF closely related to behavioral control, significantly mediated the relationship between depression severity and IED-27 factors emotional dysregulation and relationship difficulties but not non-suicidal self-injuries.ConclusionOn average, adolescents with depression show only subtle deficits in executive functioning. However, increased EF deficits are associated with the occurrence of comorbid borderline personality features, contributing to a more severe overall psychopathology. Therefore, training of executive functioning might have a positive effect on psychosocial functioning in severely depressed adolescents, as it might also improve comorbid BPF.Clinical trial registrationwww.ClinicalTrials.gov, identifier NCT03167307

The ligand-free state of the TPP riboswitch: a partially folded RNA structure

Riboswitches are elements of mRNA that regulate gene expression by undergoing structural changes upon binding of small ligands. Although the structures of several riboswitches have been solved with their ligands bound, the ligand-free states of only a few riboswitches have been characterized. The ligand-free state is as important for the functionality of the riboswitch as the ligand-bound form, but the ligand-free state is often a partially folded structure of the RNA with conformational heterogeneity that makes it particularly challenging to study. Here, we present models of the ligand-free state of a thiamine pyrophosphate (TPP) riboswitch that are derived from a combination of complementary experimental and computational modeling approaches. We obtain a global picture of the molecule using small angle X-ray scattering (SAXS) data and use an RNA structure modeling software, MC-SYM, to fit local structural details to this data on an atomic scale. We have used two different approaches to obtain these models. Our first approach develops a model of the RNA from the structures of its constituent junction fragments in isolation. The second approach treats the RNA as a single entity, without bias from the structure of its individual constituents. We find that both approaches give similar models for the ligand-free form, but the ligand-bound models differ for the two approaches, and only the models from the second approach agree with the ligand-bound structure known previously from X-ray crystallography. Our models provide a picture of the conformational changes that may occur in the riboswitch upon binding its ligand. Our results also demonstrate the power of combining experimental SAXS data with theoretical structure prediction tools in the determination of RNA structures beyond riboswitches

Xylem Phenology and Growth Response of European Beech, Silver Fir and Scots Pine along an Elevational Gradient during the Extreme Drought Year 2018

Highlights: European beech (Fagus sylvatica L.) and silver fir (Abies alba Mill.) displayed parabolic elevational trends of the cessation of xylem cell differentiation phases. Xylem phenology and growth rates of Scots pine (Pinus sylvestris L.) appeared to be less influenced by the 2018 drought, whereas beech reduced growth on the lowest elevation and fir seemed negatively affected in general. Background: The year 2018 was characterized by multiple drought periods and heat waves during the growing season. Our aim was to understand species-specific responses of xylem phenology and growth to drought and how this effect was modified along an elevational gradient. Materials and Methods: We sampled microcores and increment cores along an elevational gradient in the southwestern Black Forest (SW Germany) region and analyzed xylem phenology and growth response to drought. Results: Termination of cell enlargement and lignification occurred earliest in beech and latest in pine. Beech had the highest growth rates but shortest growth durations, fir achieved moderate rates and medium durations and pine had lowest growth rates despite long growth durations. In contrast to pine, onsets of cell differentiation phases of fir and beech did not show clear linear relationships with elevation. Cessation of cell production and lignification of beech and fir followed a parabolic elevational trend and occurred earliest on low elevations, whereas pine showed no changes with elevation. Tree-ring width, generally, depended 3–4 times more on the growth rate than on growth duration. Conclusions: The possibly drought-induced early cessation of cell differentiation and considerable growth reduction of beech appeared to be most severe on the lowest elevation. In comparison, growth reductions of fir were larger and seemed independent from elevation. We found evidence, that productivity might be severely affected at lower elevations, whereas at high elevations wood production might not equally benefit during global warming

Xylem Phenology and Growth Response of European Beech, Silver Fir and Scots Pine along an Elevational Gradient during the Extreme Drought Year 2018

Highlights: European beech (Fagus sylvatica L.) and silver fir (Abies alba Mill.) displayed parabolic elevational trends of the cessation of xylem cell differentiation phases. Xylem phenology and growth rates of Scots pine (Pinus sylvestris L.) appeared to be less influenced by the 2018 drought, whereas beech reduced growth on the lowest elevation and fir seemed negatively affected in general. Background: The year 2018 was characterized by multiple drought periods and heat waves during the growing season. Our aim was to understand species-specific responses of xylem phenology and growth to drought and how this effect was modified along an elevational gradient. Materials and Methods: We sampled microcores and increment cores along an elevational gradient in the southwestern Black Forest (SW Germany) region and analyzed xylem phenology and growth response to drought. Results: Termination of cell enlargement and lignification occurred earliest in beech and latest in pine. Beech had the highest growth rates but shortest growth durations, fir achieved moderate rates and medium durations and pine had lowest growth rates despite long growth durations. In contrast to pine, onsets of cell differentiation phases of fir and beech did not show clear linear relationships with elevation. Cessation of cell production and lignification of beech and fir followed a parabolic elevational trend and occurred earliest on low elevations, whereas pine showed no changes with elevation. Tree-ring width, generally, depended 3–4 times more on the growth rate than on growth duration. Conclusions: The possibly drought-induced early cessation of cell differentiation and considerable growth reduction of beech appeared to be most severe on the lowest elevation. In comparison, growth reductions of fir were larger and seemed independent from elevation. We found evidence, that productivity might be severely affected at lower elevations, whereas at high elevations wood production might not equally benefit during global warming