Die Bedeutung von Miro1/RHOT1 als Kandidatengen für eine gestörte mitochondriale Funktion und Dynamik bei der Parkinson-Krankheit

Die Parkinson Krankheit (PK) ist eine der häufigsten neurodegenerativen Erkrankungen des Menschen. Bei der Entstehung der Erkrankung spielen verschiedene Faktoren eine Rolle, wobei neben Umweltfaktoren auch verschiedene genetische Faktoren, für die Entstehung der PK verantwortlich sind. So können aktuell ca. 5-10% der Erkrankungen durch monogenetische Formen erklärt werden. Zudem gibt es mind. 28 weitere genetische Risikofaktoren, durch die die Wahrscheinlichkeit, die PK zu entwickeln, erhöht wird. Insgesamt lassen sich durch alle genetischen Faktoren zurzeit etwa 20-30% des Risikos, eine PK zu entwickeln, erklären. Es wird daher intensiv nach weiteren genetischen Ursachen gesucht. Hier gilt das Protein Miro1 als Kandidat, eine Rolle in der Pathogenese der PK zu spielen. Miro1 ist ein Protein der Gruppe der mitochondrialen Rho-GTPasen, das eine entscheidende Rolle im Transport von Mitochondrien in den Neuronen, sowie im Rahmen der Qualitätskontrolle der Mitochondrien spielt. Da bereits aus anderen Studien bekannt war, dass Störungen dieser Funktionen mit der Entstehung der PK assoziiert sind, haben wir im Rahmen einer Kandidatengen-Studie nach Mutationen im Miro1/RHOT1-Gen bei 752 Parkinson-Patienten gesucht. Hierfür wurden die DNA-Proben mittels High Resolution Melting Analysis, Sequenzierung, SNaPshot® und/oder Restriktionsenzymverdau untersucht. Hierbei haben wir insgesamt 10 Single Nukleotid Polymorphism (SNP) beziehungsweise Mutationen nachweisen können. Bei drei dieser SNPs handelt es sich um intronische, nicht-kodierende Basenaustausche und bei 4 weiteren SNPs um synonymous coding SNPs, also Basenaustausche, die die Peptid-Sequenz nicht ändern. Wir haben bei dieser Arbeit jedoch auch drei bisher unbekannt missense Mutationen im Miro1-Gen bei PK-Patienten nachweisen können. Diese drei Mutationen liegen in relevanten strukturellen Bereichen von Miro1 (einer ligand mimic domain an der ersten EF-Hand-Domäne, einer GTPase-Domäne und der Transmembran-Domäne). Diese Mutationen wurden zudem auch durch die Software MutationTaster als krankheitsauslösend eingestuft. Durch den Nachweis von drei bisher unbekannten Mutationen im Miro1/RHOT1-Gen bei PK-Patienten sehen wir die Bedeutung von Miro1/RHOT1 als Kandidatengen für eine gestörte mitochondriale Funktion und Dynamik bei der Parkinson-Krankheit gestärkt. Nachfolgende funktionelle Studien an Patienten-basierten Zellmodellen haben diesen Verdacht zwischenzeitlich bestätigt und weisen auf eine Störung der Calcium-Pufferkapazität der Mitochondrien durch die o.g. Mutationen hin

Thermal rectification effects of multiple semiconductor quantum dot junctions

Based on the multiple energy level Anderson model, this study theoretically examines the thermoelectric effects of semiconductor quantum dots (QDs) in the nonlinear response regime. The charge and heat currents in the sequential tunneling process are calculated by using the Keldysh Green's function technique. Results show that the thermal rectification effect can be observed in a multiple QD junction system, whereas the tunneling rate, size fluctuation, and location distribution of QD significantly influence the rectification efficiency.Comment: 5 pages, 8figure

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2; the Spanish projects PID2019-106166RB-100-OXYWAVE, PID2020-118968RB-100-LILI, PID2021-123332OB-C21 and PID2019-107098RJ-I00, funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/; the “Comunidad de Madrid” Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM; the Generalitat Valenciana projects CIPROM/2021/079-PROMETEO and SEJI/2020/007; and the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. P.G.d.S. thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and the Ministry of Science and Innovation for her contract as part of the PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR. D.G.-P. thanks Juan de la Cierva Incorporación contract Ref. No.: IJC2020-043725-I, funded by MCIN/AEI/10.13039/501100011033, and the EU NextGenerationEU/PRTR program. K.Ś. thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramón y Cajal contract (Ref. RYC2020-030596-I). We thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline

Molecular Evolution of Multiple Arylalkylamine N-Acetyltransferase (AANAT) in Fish

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to arylalkylamines, including indolethylamines and phenylethylamines. Multiple aanats are present in teleost fish as a result of whole genome and gene duplications. Fish aanat1a and aanat2 paralogs display different patterns of tissue expression and encode proteins with different substrate preference: AANAT1a is expressed in the retina, and acetylates both indolethylamines and phenylethylamines; while AANAT2 is expressed in the pineal gland, and preferentially acetylates indolethylamines. The two enzymes are therefore thought to serve different roles. Here, the molecular changes that led to their specialization were studied by investigating the structure-function relationships of AANATs in the gilthead seabream (sb, Sperus aurata). Acetylation activity of reciprocal mutated enzymes pointed to specific residues that contribute to substrate specificity of the enzymes. Inhibition tests followed by complementary analyses of the predicted three-dimensional models of the enzymes, suggested that both phenylethylamines and indolethylamines bind to the catalytic pocket of both enzymes. These results suggest that substrate selectivity of AANAT1a and AANAT2 is determined by the positioning of the substrate within the catalytic pocket, and its accessibility to catalysis. This illustrates the evolutionary process by which enzymes encoded by duplicated genes acquire different activities and play different biological roles

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential

Soft matter roadmap

Soft materials are usually defined as materials made of mesoscopic entities, often self-organised, sensitive to thermal fluctuations and to weak perturbations. Archetypal examples are colloids, polymers, amphiphiles, liquid crystals, foams. The importance of soft materials in everyday commodity products, as well as in technological applications, is enormous, and controlling or improving their properties is the focus of many efforts. From a fundamental perspective, the possibility of manipulating soft material properties, by tuning interactions between constituents and by applying external perturbations, gives rise to an almost unlimited variety in physical properties. Together with the relative ease to observe and characterise them, this renders soft matter systems powerful model systems to investigate statistical physics phenomena, many of them relevant as well to hard condensed matter systems. Understanding the emerging properties from mesoscale constituents still poses enormous challenges, which have stimulated a wealth of new experimental approaches, including the synthesis of new systems with, e.g. tailored self-assembling properties, or novel experimental techniques in imaging, scattering or rheology. Theoretical and numerical methods, and coarse-grained models, have become central to predict physical properties of soft materials, while computational approaches that also use machine learning tools are playing a progressively major role in many investigations. This Roadmap intends to give a broad overview of recent and possible future activities in the field of soft materials, with experts covering various developments and challenges in material synthesis and characterisation, instrumental, simulation and theoretical methods as well as general concepts

Osteoimmunology of Oral and Maxillofacial Diseases : Translational Applications Based on Biological Mechanisms

The maxillofacial skeleton is highly dynamic and requires a constant equilibrium between the bone resorption and bone formation. The field of osteoimmunology explores the interactions between bone metabolism and the immune response, providing a context to study the complex cellular and molecular networks involved in oro-maxillofacial osteolytic diseases. In this review, we present a framework for understanding the potential mechanisms underlying the immuno-pathobiology in etiologically-diverse diseases that affect the oral and maxillofacial region and share bone destruction as their common clinical outcome. These otherwise different pathologies share similar inflammatory pathways mediated by central cellular players, such as macrophages, T and B cells, that promote the differentiation and activation of osteoclasts, ineffective or insufficient bone apposition by osteoblasts, and the continuous production of osteoclastogenic signals by immune and local stromal cells. We also present the potential translational applications of this knowledge based on the biological mechanisms involved in the inflammation-induced bone destruction. Such applications can be the development of immune-based therapies that promote bone healing/regeneration, the identification of host-derived inflammatory/collagenolytic biomarkers as diagnostics tools, the assessment of links between oral and systemic diseases; and the characterization of genetic polymorphisms in immune or bone-related genes that will help diagnosis of susceptible individuals.Peer reviewe

Virulenz von aviären Influenzaviren des Subtypes H7 in Vögeln: Der Einfluss der polybasischen Spaltstelle im Hämagglutinin auf die Virulenz eines aktuellen deutschen H7N7 Virus und die Pathogenität von europäischen H7N7 und H7N1 Viren in Enten

Avian influenza viruses of subtype H7Nx cause high economic losses and can evolve, similar to H5Nx viruses, to highly pathogenic viruses from low pathogenic ancestors. Some H7Nx viruses are endemic in Europe and are frequently reported to cause outbreaks in different poultry species. While turkeys and chickens are highly susceptible as indicated by high mortality, ducks are mostly clinically resistant to avian influenza virus (AIV). In 2015 a natural pair of low pathogenic AIV (LPAIV)/ high pathogenic AIV (HPAIV) H7N7 virus was isolated from the same farm in the district Emsland, North West Germany. The genetic comparison between the LP and the HP H7N7 virus showed mutations in all eight segments and a polybasic cleavage site motif in the HP virus leading to high virulence and 100% mortality in chickens. In this thesis virulence determinants of this virus were investigated by reverse genetics and infection experiments in chickens, turkeys and ducks. In the first publication we focused on the impact of the polybasic cleavage site of the H7N7 virus as a main virulence determinant in different bird species. While the polybasic cleavage site increased virulence and endothelial tropism of LPAIV in chickens to levels similar to the HP virus, it was less important but still necessary for high virulence in turkeys, but not in ducks. Pekin, Mallard and Muscovy ducks were susceptible to infection with HPAIV H7N7, but only Muscovy ducks showed mild to moderate clinical signs. Since Mallard ducks shed remarkable amounts of virus, they may play a role in the silent spread of HPAIV H7 viruses. In the second publication, we assessed virulence of different European H7 viruses in Muscovy and Pekin ducks. In Muscovy ducks, the Fowl Plague Virus (FPV) H7N7 from 1927 caused 20% mortality, while the two H7N1 viruses from 1934 from Germany and from 1999 from Italy showed moderate to high virulence with a mortality rate ranging from 20 to 80% after intrachoanal inoculation. Although all viruses were highly virulent in chickens and possess a polybasic CS they exhibit variable virulence in ducks. These findings show the importance of the investigation of virulence determinants of H7 viruses in ducks. Taken together, virulence determinants and pathobiology of HPAIV H7 is different according to the bird species. In chickens, the polybasic CS is the major determinant for virulence and tropism, while other gene segments are important for virulence and transmission in turkeys. Chickens and turkeys are dead-end hosts playing minimal role in the spread of HPAIV, whereas asymptomatically infected ducks can further spread the virus. These findings are crucial to understand the molecular biology of HPAIV in different bird species.Aviäre Influenzviren (AIV) des Subtypes H7Nx verursachen große wirtschaftliche Schäden und können sich, genau wie H5Nx Viren, zu hoch virulenten Viren aus niedrig pathogenen Vorläufern entwickeln. Einige H7Nx Viren sind endemisch in Europa und führen regelmäßig zu Ausbrüchen in verschieden Geflügelspezies. Während Hühner und Puten sehr empfänglich sind, was durch eine hohe Mortalitätsrate gekennzeichnet ist, sind Enten meistens klinisch resistent gegen aviäre Influenzaviren. 2015 konnte ein natürliches Paar niedrig- (LP) und hochpathogener (HP) Phänotypen eines H7N7 Virus von einem Geflügelbetrieb im Landkreis Emsland, im Nordwesten Deutschlands, isoliert werden. Der genetische Vergleich beider Viren zeigte Mutationen in allen acht Segmenten und eine polybasische Spaltstelle im HP Virus, welche zu hoher Virulenz und 100% Mortalität in Hühnern führten. In dieser Arbeit wurden die Virulenzdeterminanten dieser Viren mithilfe reverser Genetik und Infektionsversuchen in Hühnern, Puten und Enten untersucht. In der ersten Publikation lag das Augenmerk auf dem Einfluss der polybasischen Spaltstelle des H7N7 Viruses als Hauptvirulenzdeterminante in verschiedenen Vogelspezies. Während die polybasische Spaltstelle die Virulenz und den Endotheltropismus des niedrigpathogenen aviären Influenzaviruses (LPAIV) in Hühnern auf vergleichbare Werte wie das hochpathogene (HP) Virus erhöhte, war sie weniger wichtig, aber dennoch für hohe Virulenz in Puten notwendig, jedoch nicht in Enten. Peking-, Stock- und Moschusenten sind empfänglich für die Infektion mit HP H7N7, aber nur die Moschusenten zeigten ein mildes bis moderates Krankheitsbild. Da Stockenten beachtenswerte Mengen des Virus ausschieden, könnten sie eine Rolle als stille Überträger von hochpathogenen aviären Influenzaviren (HPAIVs) des Subtyps H7 spielen. In der zweiten Publikation haben wir die Virulenz verschiedener europäischer H7 Viren in Moschus- und Pekingenten ermittelt. In Moschusenten verursachte das Fowl Plague Virus (FPV) H7N7 von 1927 eine Mortalität von 20%, während die beiden H7N1 Viren von 1934 aus Deutschland und von 1999 aus Italien moderate bis hohe Virulenz mit einer Mortalität von 20 bis 80% nach intrachoanaler Inokulation zeigten. Obwohl alle Viren hochvirulent in Hühnern waren und eine polybasische Spaltstelle aufwiesen, führten sie zu variabler Virulenz in Enten

A ray model for head waves in a fluid-filled borehole

A model, suggested by tbe ray expansion of Roever et al., is constructed to rapidly generate the compressional and shear refracted arrivals, known as head waves, received from a point source on the axis of an ideal fluid-filled borehole. An impulse response is derived, and its frequency characteristics are investigated. The waves are compared to those obtained by the real axis integration method of Tsang and Rader, which results in a complete waveform, including the modes as well as the refracted arrivals. The ray model gives accurate results for the compressional head wave. The shear region of the complete waveform contains strong modal interference, making it difficult to evaluate the quality of the ray model shear wave. A useful filter results from insight provided by the basic structure of the model. This filter can be used to estimate the borehole diameter or formation compressional velocity. It can also remove the second and later compressional arrivals, thus providing an accurate estimate of the source pulse and and a relatively uncorrupted vie of the initial arrival in the shear region