In Silico Studies on Proteins for Synthetic Biology

Synthetic biology develops artificial biomolecules or biological systems with novel functionalities for diverse applications in research, medicine or industry. This thesis focuses on in silico studies of three proteins that are promising candidates for enzymatic plastic waste treatment and highly sensitive biosensors, respectively. The first candidate is the enzyme Fusarium solani Cutinase (Longhi and Cambillau 1999), which is able to degrade synthetic polymers, like PET. It allows for the development of an environmental friendly and sustainable solution for plastic waste treatment on an industrial scale. As the wildype enzyme loses its activity during the process of PET degradation, a rational design approach was followed, to improve the activity of this enzyme for PET as substrate. Via MD simulations and linear response theory (LRT) (Ikeguchi et al. 2005) based on coarse-grained elastic network models, the reason for the loss of activity could be identified. Based on the knownledge gained, mutants with improved activity for PET were proposed. In the context of this study, an extension for the LRT method similar to that of a previous study (Knorr 2015) was developed. The second protein system, the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel (Santoro and Tibbs 1999), regulates the flux of ions across biological membranes by changes in the membrane voltage and binding of the ligand cAMP. Hence, it is an ideal model for studying the interplay of different domains during the gating process. Together with plenty of other ion channels, it can also serve as building blocks for the assembly of different domains to design synthetic ion channels with novel functionalities. To understand the complex mechanism of HCN gating, the extension of the LRT method was adjusted to work for a tetramer and was used to determine the conformational changes that occur upon binding of the ligand cAMP. In this context, movements in the transmembrane domains that are involved in the gating process were discovered for the first time. They provide important information on the complex gating mechanism and enable a directed planning of further experimental and theoretical investigations. Small viral pore forming proteins also enable the flux of ions across biological membranes and therefore can be seen as viral companions of ion channels. The third protein is such a pore forming protein from HIV and simian relatives SIV, called Vpu (Cohen et al. 1988). As this small protein is less complex than ion channels but also exhibits ion channel function, it is another candidate to serve as building block for the design of artificial ion channels. To consider the Vpu protein as possible building block, the formation of an ion conducting pore has to be a reliable property. In this thesis, the evolutionary conservation of ion channel formation was proved by computing the Shannon entropy (Strait and Dewey 1996) for involved residues based on a multiple sequence alignment. Although the study could not clarify the role of the ion channel function for virus release or replication, the detected evolutionary conservation serves as proof for the functional significance. Hence, this protein reliably forms an ion conducting pore and can be further considered as possible building block for the assembly of synthetic ion channels

Individuelles neuro-auditorisches Profil von Kindern mit ADHS

Die Aufmerksamkeitsdefizit-Hyperaktivitätsstörung (AD(H)S) ist eine der häufigsten psychiatrischen Diagnosen des Kindes-und Jugendalters mit einer weltweiten Prävalenz von ca. 5 %. Sie ist eine chronisch verlaufende Verhaltensstörung im Kindes-und Jugendalter und manifestiert sich mit einer Wahrscheinlichkeit von 30-50% bei veränderter Symptomatik (Aufmerksamkeits-und Konzentrationsstörungen treten in den Vordergrund, während die Hyperaktivität als sichtbares äußerliches Verhalten einem Gefühl der inneren Unruhe weicht) bis ins Erwachsenenalter. Die Diagnose erfolgt mehrheitlich auf der Basis von Verhaltensbeobachtungen, was zu diagnostischen Unterschieden zwischen Institutionen, Kulturen und Ländern führt. Eine objektive, auf Gehirndaten basierende Diagnose ist noch nicht etabliert. AD(H)S kann entweder durch das DSM oder ICD diagnostiziert werden. Die diagnostischen Kriterien und die Validität der Subtypen ADHS und ADS haben sich mit jeder neuen Ausgabe des DSM und ICD verändert und sind immer Grundlage der Diskussionsdebatte. Es ist noch wenig über den Einfluss von musikalischer Erfahrung auf die anatomische und funktionelle Entwicklung des auditorischen Cortex bei Kindern mit Entwicklungsstörungen wie AD(H)S bekannt. In dieser Doktorarbeit wurden in einer Querschnittsstudie die neuroanatomischen und neurofunktionellen Profile des auditorischen Cortex, ebenso wie die auditorische Leistungsfähigkeit von 37 Kindern mit diagnostizierter Aufmerksamkeitsdefizit/-Hyperaktivitätsstörung (Subtyp ADHS) mit einer Kontrollgruppe in Bezug auf die Intensität der musikalischen Praxis, die musikalische Begabung und die musikalische Performanz verglichen. Die Fragestellungen dieser Doktorarbeit beziehen sich auf die strukturellen und funktionellen Eigenschaften des auditorischen Cortex, auf die auditorisch-kognitiven Fähigkeiten und auf die Auswirkung des intensiven Musizierens auf die auditorisch-kognitiven Fähigkeiten bei Kindern mit dem Subtyp ADHS. Auf Grund der dargestellten Ergebnisse der AMseL II-Studie wurde festgestellt, dass es ein spezifisches auditorisches und neurologisches Profil von Kindern mit dem Subtyp ADHS gibt, das sich sowohl vom Subtyp ADS als auch von dem der Kontrollgruppe unterscheidet. Es stellt sich hier aber weiterhin die Frage, ob dieses spezielle auditorische Profil bei Kindern mit dem Subtyp ADHS auch mit speziellen auditorischen Eigenschaften verbunden ist. In der AMseL I-Studie zeigten Kinder mit AD(H)S verkleinerte Volumen des HG, kleinere HG/ PT-Verhältnisse und vergrößerte Volumen des PT, die mit einer bilateralen P1-Asynchronizität einhergingen. In dieser vorausgegangenen Studie (Seither-Preisler 2014) wurde keine Unterscheidung zwischen den Subtypen ADHS und ADS gemacht, es zeigte sich aber in Pilotstudien, dass sich das neuro-auditorische Profil von ADS und ADHS deutlich unterscheiden müsste. Daraus folgte die Annahme, dass Kinder mit dem Subtyp ADHS ein eigenes, spezifisches neuroanatomisches und neurofunktionelles auditorisches Profil zeigen müssten: bei ADHS größere PTs, verkleinerte HGs und daraus resultierend kleinere HG/ PT-Verhältnisse, sowie bilateralen P1-Asynchronizitäten, bei ADS hingegen im rechten AC völlig andere anatomische Biomarker als bei ADHS (Serrallach, Groß et al., 2016). Es wurde in der vorliegenden Arbeit angenommen, dass musizierende Kinder mit ADHS größere Volumen der grauen Substanz des ACs haben als nicht musizierende Kinder mit ADHS. Ebenso sollten musikalisch trainierte Kinder mit dem Subtyp ADHS bessere Ergebnisse in den auditorischen Tests erzielen als nicht musikalische Kinder mit ADHS. Kinder mit der Diagnose ADHS zeigten verglichen mit nicht betroffenen Gleichaltrigen der Kontrollgruppe eine charakteristische Morphologie im auditorischen Cortex mit einem kleineren linken HG, einem vergrößerten linken und rechten PT, und daraus resultierend einem beidseitig kleineren HG/ PT-Verhältnis. Weiterhin konnte bei Kindern mit dem Subtyp ADHS eine deutlich vergrößerte bilaterale P1-Asynchronizität mit einer verspäteten P1-Antwort links und einer verfrühten P1-Antwort rechts im Vergleich zur Kontrollgruppe gefunden werden. Die Leistung der Kinder mit dem Subtyp ADHS unterschieden sich auch in den auditorisch-kognitiven Messungen von der Kontrollgruppe. Kinder der Kontrollgruppe erzielten im rhythmischen Wert und im Gesamtwert des IMMA- Tests und im Gesamtwert und in der Reaktionszeit des Metric Tests deutlich bessere Ergebnisse als die Kinder mit ADHS. Musizierende Kinder mit ADHS konnten Frequenzen im Dinosaurier-Test besser erkennen und hatten einen besseren IMMA-Gesamtwert als die nicht musizierenden Kinder mit ADHS. Ebenso zeigten sich einige Zusammenhänge zwischen dem intensiven Musizieren und den neurologischen Besonderheiten und den auditorisch-kognitiven Fähigkeiten der Kinder mit dem Subtyp ADHS. Kinder mit ADHS mit einer kleineren Latenzdifferenz |R-L| zeigten kleinere Inkonsistenzwerte im Pitch-Test und kleinere Frequenzunterschiede im Dino-Test. Kinder mit ADHS und einem größeren rechten Verhältnis (HG/PT) zeigten bessere Werte in der Rhythmuserkennung und konnten kleinere Lautstärkeunterschiede wahrnehmen. Kinder mit ADHS mit einem größeren linken HG zeigten ein besseres musikalisches Gedächtnis und eine bessere Rhythmuserkennung im Musiziertest. Kinder mit einem größeren linken HG/PT-Verhältnis zeigten ebenfalls bessere Werte im Rhythmustest. Die Effekte waren in der linken Hemisphäre deutlich stärker ausgeprägt als in der rechten Hemisphäre. In der Diskussion wird bezugnehmend auf meine mit Frau Serrallach gemeinsam und als gleichberechtigte Erstautorin geschriebene Publikation (Serrallach, Groß et al., 2016) darauf hingewiesen, dass es möglich ist, ein spezifisches neuro-auditorisches Profil von Kindern mit dem Subtyp ADHS zu identifizieren, das sich von der Kontrollgruppe (Doktorarbeit C. Groß) und von der ADS-Gruppe (Doktorarbeit B. Serrallach) mit einer Sensitivität von ~90 % abgrenzt. In diesem Sinne erlauben es die identifizierten neurologischen Biomarker, eine spezifische Differentialdiagnose für ADHS in Abgrenzung zu ADS zu erstellen (Serrallach, Groß et al., 2016; Seither- Preisler et al., 2014). Diese Doktorarbeit zeigt, dass ein größeres linkes PT und ein verkleinertes linkes HG/PT-Verhältnis die ADHS- von der Kontrollgruppe unterscheidet. In Bezug auf den rechten AC kann die ADHS-Gruppe darüber hinaus von der ADS-Gruppe unterschieden werden. Außerdem zeigen Kinder mit ADHS und ADS eine bilaterale Asynchronizität, die meistens linksseitig verspätet ist und bei Kindern mit ADHS im Gegensatz zu Kindern mit ADS auffällige L-R-Asymmetrien bzgl. der Amplitude und Breite der P1-Antwort aufweist (Serrallach, Groß et al., 2016). Zusammenfassend ist es möglich, durch die bilaterale P1-Asynchronizität und die Größe und das Verhältnis des linken AC in einem ersten Schritt zwischen AD(H)S und der Kontrollgruppe zu unterscheiden und in einem zweiten Schritt aufgrund der rechtshemisphärischen Unterscheide eine Differentialdiagnose zwischen ADHS und ADS zu erstellen (Serrallach, Groß et al, 2016). In diesem Sinne können neuroanatomische und neurofunktionelle Parameter des auditorischen Cortex dazu beitragen, zusätzliche wertvolle Kennzeichen für ein zukünftiges auf Gehirndaten basierendes objektives Diagnoseverfahren für auditorisch bezogene Entwicklungsstörungen im Generellen und im Speziellen für die Subgruppen ADHS und ADS zu definieren. Daraus resultierend ist es notwendig, weiterführend und detailliert zu forschen, um diese auditorisch evozierten Besonderheiten bei Kindern mit ADHS und ADS genauer zu eruieren und auf Basis der gezeigten musizierbedingten Neuroplastizität bei Kindern mit ADHS eine speziell auf das individuelle neuro-auditorische Profil abgestimmte musikalisch basierte Therapie zu entwickeln

Misjudgement of One’s Own Performance? Exploring Attention Deficit (Hyperactivity) Disorder (ADHD) and Individual Difference in Complex Music and Foreign Language Perception

In previous research, we detected that children and adolescents who were diagnosed with ADHD showed deficits in both complex auditory processing of musical stimuli and in musical performance when compared to controls. In this study, we were interested in whether we could detect similar or distinct findings when we use foreign speech perception tasks. Therefore, we recruited musically naïve participants (n = 25), music-educated participants (n = 25) and participants diagnosed with ADHD (n = 25) who were assessed for their short-term memory (STM) capacity and the ability to discriminate music and speech stimuli and we collected self-ratings of the participants’ music and language performances. As expected, we found that young adults with ADHD show deficits in the perception of complex music and difficult speech perception stimuli. We also found that STM capacity was not impaired in young adults with ADHD and may not persist into young adulthood. In addition, subjective self-estimation about the participants’ language and music performances revealed that the ADHD group overestimated their performance competence relatively compared to both control groups. As a result, the findings of our study suggest that individuals diagnosed with ADHD require a special training program that not only focuses on improving performance in perceptual skills of music and language but also requires metacognitive training to develop realistic self-assessment skills

Identification of semester-specific teaching contents for dental ethics: development, testing and validation of a questionnaire

Background Although medical ethics is an indispensable part of dental education, it has not played a relevant role in the dental curriculum thus far. This study is aimed at developing and validating a questionnaire that identifies semester-specific ethical issues, in order to develop longitudinal ethic modules. Methods March 2018 a workshop on item generation was coordinated, using Delphi method; followed by a cognitive testing with students (2nd, 4th, 10th semesters, n = 12). A pilot test was carried out with students from different semesters (n = 60). The distribution of response frequencies and missing values were determined. The questionnaire used for validation consisted of three dimensions: ethical knowledge, dealing with ethical issues, expectations in terms of teaching. The psychometric examination was carried out by preclinical students (n = 105) and clinical semesters (n = 110) January 2019. Results After cognitive testing and piloting, some items were reformulated, so that a questionnaire with 127 items was used for validation. The individual dimensions were assigned to various factors with excellent to acceptable internal consistency (Cronbach's α 0.72-0.96). Conclusion The questionnaire has an acceptable to excellent consistency and suggests that the different dimensions are conclusive. With this questionnaire, ethical issues in dentistry can be mapped and teaching contents identified

Musical Performance in Adolescents with ADHD, ADD and Dyslexia—Behavioral and Neurophysiological Aspects

Research has shown that dyslexia and attention deficit (hyperactivity) disorder (AD(H)D) are characterized by specific neuroanatomical and neurofunctional differences in the auditory cortex. These neurofunctional characteristics in children with ADHD, ADD and dyslexia are linked to distinct differences in music perception. Group-specific differences in the musical performance of patients with ADHD, ADD and dyslexia have not been investigated in detail so far. We investigated the musical performance and neurophysiological correlates of 21 adolescents with dyslexia, 19 with ADHD, 28 with ADD and 28 age-matched, unaffected controls using a music performance assessment scale and magnetoencephalography (MEG). Musical experts independently assessed pitch and rhythmic accuracy, intonation, improvisation skills and musical expression. Compared to dyslexic adolescents, controls as well as adolescents with ADHD and ADD performed better in rhythmic reproduction, rhythmic improvisation and musical expression. Controls were significantly better in rhythmic reproduction than adolescents with ADD and scored higher in rhythmic and pitch improvisation than adolescents with ADHD. Adolescents with ADD and controls scored better in pitch reproduction than dyslexic adolescents. In pitch improvisation, the ADD group performed better than the ADHD group, and controls scored better than dyslexic adolescents. Discriminant analysis revealed that rhythmic improvisation and musical expression discriminate the dyslexic group from controls and adolescents with ADHD and ADD. A second discriminant analysis based on MEG variables showed that absolute P1 latency asynchrony |R-L| distinguishes the control group from the disorder groups best, while P1 and N1 latencies averaged across hemispheres separate the control, ADD and ADHD groups from the dyslexic group. Furthermore, rhythmic improvisation was negatively correlated with auditory-evoked P1 and N1 latencies, pointing in the following direction: the earlier the P1 and N1 latencies (mean), the better the rhythmic improvisation. These findings provide novel insight into the differences between music processing and performance in adolescents with and without neurodevelopmental disorders. A better understanding of these differences may help to develop tailored preventions or therapeutic interventions

Phosphatidylserine-exposing extracellular vesicles in body fluids are an innate defence against apoptotic mimicry viral pathogens

Some viruses are rarely transmitted orally or sexually despite their presence in saliva, breast milk, or semen. We previously identified that extracellular vesicles (EVs) in semen and saliva inhibit Zika virus infection. However, the antiviral spectrum and underlying mechanism remained unclear. Here we applied lipidomics and flow cytometry to show that these EVs expose phosphatidylserine (PS). By blocking PS receptors, targeted by Zika virus in the process of apoptotic mimicry, they interfere with viral attachment and entry. Consequently, physiological concentrations of EVs applied in vitro efficiently inhibited infection by apoptotic mimicry dengue, West Nile, Chikungunya, Ebola and vesicular stomatitis viruses, but not severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus 1, hepatitis C virus and herpesviruses that use other entry receptors. Our results identify the role of PS-rich EVs in body fluids in innate defence against infection via viral apoptotic mimicries, explaining why these viruses are primarily transmitted via PS-EV-deficient blood or blood-ingesting arthropods rather than direct human-to-human contact

School-based mental health promotion in children and adolescents with StresSOS using online or face-to-face interventions: study protocol for a randomized controlled trial within the ProHEAD Consortium

Abstract Background Schools are an ideal setting in which to promote health. However, empirical data on the effectiveness of school-based mental health promotion programs are rare, and research on universal Internet-based prevention in schools is almost non-existent. Following the life skills approach, stress management training is an important component of health promotion. Mental health literacy is also associated with mental health status, and it facilitates formal help-seeking by children and adolescents (C&A). The main objectives of this study are (1) the development and evaluation of an Internet-based version of a universal school-based health promotion program called StresSOS and (2) demonstrating non-inferiority of the online setting compared to the face-to-face setting. StresSOS aims to improve stress management and mental health literacy in C&A. Methods/design A school-based sample of 15,000 C&A (grades 6–13 and older than 12 years) will be recruited in five regions of Germany within the ProHEAD Consortium. Those with a screening result at baseline indicating no mental health problems will be invited to participate in a randomized controlled trial comparing StresSOS online to an active online control condition (Study A). In addition, 420 adolescents recruited as a separate school-based sample will participate in the StresSOS face-to-face intervention. Participants in both intervention groups (online or face-to-face) will receive the same eight treatment modules to allow for the comparison of both methods of delivery (Study B). The primary outcome is the number of C&A with symptoms of mental health problems at a 12 months follow-up. Secondary outcomes are related to stress/coping (i.e., knowledge, symptoms of stress, coping resources), mental health literacy (knowledge and attitudes toward mental disorders and help-seeking), program usage patterns, cost-effectiveness, and acceptability of the intervention. Discussion This study represents the first adequately powered non-inferiority trial in the area of school-based mental health promotion. If online StresSOS proves efficacious and non-inferior to face-to-face delivery, this offers great potential for health promotion in youths, both in and outside the school environment. Trial registration German Clinical Trials Register, DRKS00014693 . Registered on 14 May 2018

Improved upper limb function in non-ambulant children with SMA type 2 and 3 during nusinersen treatment: a prospective 3-years SMArtCARE registry study

Background The development and approval of disease modifying treatments have dramatically changed disease progression in patients with spinal muscular atrophy (SMA). Nusinersen was approved in Europe in 2017 for the treatment of SMA patients irrespective of age and disease severity. Most data on therapeutic efficacy are available for the infantile-onset SMA. For patients with SMA type 2 and type 3, there is still a lack of sufficient evidence and long-term experience for nusinersen treatment. Here, we report data from the SMArtCARE registry of non-ambulant children with SMA type 2 and typen 3 under nusinersen treatment with a follow-up period of up to 38 months. Methods SMArtCARE is a disease-specific registry with data on patients with SMA irrespective of age, treatment regime or disease severity. Data are collected during routine patient visits as real-world outcome data. This analysis included all non-ambulant patients with SMA type 2 or 3 below 18 years of age before initiation of treatment. Primary outcomes were changes in motor function evaluated with the Hammersmith Functional Motor Scale Expanded (HFMSE) and the Revised Upper Limb Module (RULM). Results Data from 256 non-ambulant, pediatric patients with SMA were included in the data analysis. Improvements in motor function were more prominent in upper limb: 32.4% of patients experienced clinically meaningful improvements in RULM and 24.6% in HFMSE. 8.6% of patients gained a new motor milestone, whereas no motor milestones were lost. Only 4.3% of patients showed a clinically meaningful worsening in HFMSE and 1.2% in RULM score. Conclusion Our results demonstrate clinically meaningful improvements or stabilization of disease progression in non-ambulant, pediatric patients with SMA under nusinersen treatment. Changes were most evident in upper limb function and were observed continuously over the follow-up period. Our data confirm clinical trial data, while providing longer follow-up, an increased number of treated patients, and a wider range of age and disease severity

In Silico Studies on Proteins for Synthetic Biology

Synthetic biology develops artificial biomolecules or biological systems with novel functionalities for diverse applications in research, medicine or industry. This thesis focuses on in silico studies of three proteins that are promising candidates for enzymatic plastic waste treatment and highly sensitive biosensors, respectively. The first candidate is the enzyme Fusarium solani Cutinase (Longhi and Cambillau 1999), which is able to degrade synthetic polymers, like PET. It allows for the development of an environmental friendly and sustainable solution for plastic waste treatment on an industrial scale. As the wildype enzyme loses its activity during the process of PET degradation, a rational design approach was followed, to improve the activity of this enzyme for PET as substrate. Via MD simulations and linear response theory (LRT) (Ikeguchi et al. 2005) based on coarse-grained elastic network models, the reason for the loss of activity could be identified. Based on the knownledge gained, mutants with improved activity for PET were proposed. In the context of this study, an extension for the LRT method similar to that of a previous study (Knorr 2015) was developed. The second protein system, the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel (Santoro and Tibbs 1999), regulates the flux of ions across biological membranes by changes in the membrane voltage and binding of the ligand cAMP. Hence, it is an ideal model for studying the interplay of different domains during the gating process. Together with plenty of other ion channels, it can also serve as building blocks for the assembly of different domains to design synthetic ion channels with novel functionalities. To understand the complex mechanism of HCN gating, the extension of the LRT method was adjusted to work for a tetramer and was used to determine the conformational changes that occur upon binding of the ligand cAMP. In this context, movements in the transmembrane domains that are involved in the gating process were discovered for the first time. They provide important information on the complex gating mechanism and enable a directed planning of further experimental and theoretical investigations. Small viral pore forming proteins also enable the flux of ions across biological membranes and therefore can be seen as viral companions of ion channels. The third protein is such a pore forming protein from HIV and simian relatives SIV, called Vpu (Cohen et al. 1988). As this small protein is less complex than ion channels but also exhibits ion channel function, it is another candidate to serve as building block for the design of artificial ion channels. To consider the Vpu protein as possible building block, the formation of an ion conducting pore has to be a reliable property. In this thesis, the evolutionary conservation of ion channel formation was proved by computing the Shannon entropy (Strait and Dewey 1996) for involved residues based on a multiple sequence alignment. Although the study could not clarify the role of the ion channel function for virus release or replication, the detected evolutionary conservation serves as proof for the functional significance. Hence, this protein reliably forms an ion conducting pore and can be further considered as possible building block for the assembly of synthetic ion channels

Probabilistic temperature forecasting with a heteroscedastic autoregressive ensemble postprocessing model

Möller AC, Groß J. Probabilistic temperature forecasting with a heteroscedastic autoregressive ensemble postprocessing model. Quarterly Journal of the Royal Meteorological Society. 2020;146(726):211-224.To account for uncertainty in numerical weather prediction (NWP) models it has become common practice to employ ensembles of NWP forecasts. However, forecast ensembles often exhibit forecast biases and dispersion errors, thus require statistical postprocessing to improve reliability of the ensemble forecasts.<br>This work proposes an extension of a recently developed postprocessing model for temperature utilizing autoregressive information present in the forecast error of the raw ensemble members. The original approach is modified to let the variance parameter additionally depend on the ensemble spread, yielding a two-fold heteroscedastic model. Furthermore, a high-resolution forecast is included into the postprocessing model, yielding improved predictive performance. Finally, it is outlined how the autoregressive model can be utilized to postprocess ensemble forecasts with higher forecast horizons, without the necessity of making fundamental changes to the original model. To illustrate the performance of the heteroscedastic extension of the autoregressive model, and its use for higher forecast horizons we present a case study for a data set containing 12 years of temperature forecasts and observations over Germany. The case study indicates that the autoregressive model yields particularly strong improvements for forecast horizons beyond 24 hours ahead