Exploring the use of a blue pigment-producing NRPS as a tagging method to easily detect engineered NRPs

Nonribosomal peptide synthetases (NRPSs) are modular mega-enzymes found in bacteria and fungi that produce nonribosomal peptides (NRPs) in an assembly line fashion. Each module is in charge of adding a specific amino acid (AA) to the growing peptide chain. Three basic domains constitute one NRPS module: the adenylation (A), peptidyl carrier protein (PCP) and condensation (C) domains. The A domain recognizes and activates the AA. An external enzyme, the PPtase, attaches a phosphopantetheine (PPant) arm to the PCP domain which then picks up the activated AA and delivers it to the C domain. The C domain recognizes the growing peptide chain (donor) as well as the new AA (acceptor) and fuses the two together. A special feature of NRPSs is their ability to recognize and incorporate not only proteinogenic AAs, but also other building blocks like fatty acids (FAs) or non-proteinogenic AAs. All building blocks can be further modified through the action of additional domains: epimerization (E), methylation (M) and oxidation (Ox) domains, among others. In this manner a great variety of different NRPs can be synthesized, many of which are bioactive and exhibit anti-microbial or anti-cancer properties. Thus, it is highly desirable to understand how NRPS domains and modules function and find ways to genetically re-engineer them for custom NRP production. Since the discovery of NRPSs, many efforts have already been made to engineer these enzymes in order to create custom NRPs, but general design rules yet remain elusive. The successful attempts to re-create functional NRPS for the production of novel NRPs include: (i) mutations of the A domain, (ii) subdomain modifications and (iii) rearrangements on the module level. Yet, many engineered NRPSs exhibit only slow reaction rates and low product yields. In some cases, the desired NRP products cannot be detected at all, possibly due to additional control mechanisms that have not been taken into account during the engineering process, such as substrate specificity of C domains. Hence there is still a great need to identify the general rules for successful NRPS engineering in order to exploit the ever-growing molecular toolbox of newly discovered NRPSs for recombinant production of novel bioactive compounds. In this work I present my attempts to develop an approach to easily monitor the outcome of NRPS manipulation using a pigment-producing synthetase as a genetic tag. To this end, I first investigated two homologous synthetases, IndC and BpsA, which produce the blue pigment indigoidine, and mutants thereof and revised the proposed biosynthesis mechanism. I then created a series of fusion constructs between modules coming from different NRPSs and IndC/BpsA to test if indigoidine-tagged peptides could be produced. I identified a promising construct for which point mutations in the upstream module resulted in weaker or null pigment production. However, the expected indigoidine-tagged AA was not detectable, which could be due to the fact that indigoidine production inevitably leads to the separation of the donor AA. These results raised further questions as to whether in a native NRPS, the same modifications lead to congruent effects in neighboring modules. I addressed this question using a fragment of a non-engineered NRPS to monitor the activity of the native and modified versions in an in vitro assay, which I present in the last part of the results. Surprisingly, the effects of the same set of modifications on neighboring modules did not only differ between the engineered NRP-pigment synthetase and the native NRPS, but also between different modules within the native NRPS. These results hint at highly individual behavior of NRPS modules, depending on the context they are in

Bevorratungsdüngung mit Schafwolle bei Bio-Frühjahrsblühern im Topf

Schafwolle wird im Zierpflanzenanbau als organischer Dünger eingesetzt. Bei der Erzeugung von Frühjahrsblühern (Bellis perennis und Myosotis sylvatica) wurde die vollständige Bevorratungsdüngung mit Schafwollpellets in drei verschiedenen Größen und mit geschnittenem Schafwollvlies getestet. Mit einer Düngungshöhe von 6-10 kg Schafwolle/m³  bei B. perennis und 10 kg/m³ bei M. sylvatica konnten gute Pflanzenqualitäten erzielt werden. Düngung mit kleinen Pellets führte zum höchsten Nmin-Gehalt im Substrat und zum besten Pflanzenwachstum, welches durch die Messung von Frischmasse, Höhe und Durchmesser der Pflanzen ermittelt wurde. Pflanzengesundheit und Gesamteindruck wurden bei Düngung mit geschnittenem Schafwollvlies am besten bewertet

Das Brandungskonglomerat der Nordsee im Steinbruch Mammendorf (Sachsen-Anhalt)

Im Steinbruch Mammendorf in Sachsen-Anhalt, Deutschland, sind konglomeratische Transgressionssedimente der paläogenen Nordsee (Latdorfium) erschlossen, die bankige Sandsteinfolgen und Andesite des Karbons und Perms überlagern. Das Konglomerat besteht aus kantengerundeten bis gerundeten Gerölle aus Andesit, anderen Vulkaniten, Sand- und Schluffsteinen, die von verfestigten teils karbonatischen Zwischenfüllungen aus Sand und Fossilbruchstücken verbacken sind. Das Korngrößenspektrum erwies sich nach Messungen als breit gestreut.In the Mammendorf quarry in Saxony-Anhalt, Germany, conglomeratic transgressional sediments of the Palaeogene North Sea (Latdorfian) are developed, overlying banked sandstone sequences and andesites of the Carboniferous and Permian. The conglomerate consists of edge-rounded to rounded boulders of andesite, other volcanic rocks, sandstones and siltstones cemented by a consolidated partly carbonate- bearing infill of sand and fossil fragments. The grain size spectrum proved to be broadly distributed according to measurement

TMEM16A and TMEM16B modulate pheromone-evoked action potential firing in mouse vomeronasal sensory neurons

The mouse vomeronasal system controls several social behaviors. Pheromones and other social cues are detected by sensory neurons in the vomeronasal organ (VNO). Stimuli activate a transduction cascade that leads to membrane potential depolarization, increase in cytosolic Ca2+ level, and increased firing. The Ca2+-activated chloride channels TMEM16A and TMEM16B are co-expressed within microvilli of vomeronasal neurons, but their physiological role remains elusive. Here, we investigate the contribution of each of these channels to vomeronasal neuron firing activity by comparing wild-type (WT) and knock-out (KO) mice. Performing loosepatch recordings from neurons in acute VNO slices, we show that spontaneous activity is modified by Tmem16a KO, indicating that TMEM16A, but not TMEM16B, is active under basal conditions. Upon exposure to diluted urine, a rich source of mouse pheromones, we observe significant changes in activity. Vomeronasal sensory neurons (VSNs) from Tmem16a cKO and Tmem16b KO mice show shorter interspike intervals (ISIs) compared with WT mice, indicating that both TMEM16A and TMEM16B modulate the firing pattern of pheromone-evoked activity in VSNs

Special topic: The association between pulse ingredients and canine dilated cardiomyopathy: addressing the knowledge gaps before establishing causation.

In July 2018, the Food and Drug Administration warned about a possible relationship between dilated cardiomyopathy (DCM) in dogs and the consumption of dog food formulated with potatoes and pulse ingredients. This issue may impede utilization of pulse ingredients in dog food or consideration of alternative proteins. Pulse ingredients have been used in the pet food industry for over 2 decades and represent a valuable source of protein to compliment animal-based ingredients. Moreover, individual ingredients used in commercial foods do not represent the final nutrient concentration of the complete diet. Thus, nutritionists formulating dog food must balance complementary ingredients to fulfill the animal's nutrient needs in the final diet. There are multiple factors that should be considered, including differences in nutrient digestibility and overall bioavailability, the fermentability and quantity of fiber, and interactions among food constituents that can increase the risk of DCM development. Taurine is a dispensable amino acid that has been linked to DCM in dogs. As such, adequate supply of taurine and/or precursors for taurine synthesis plays an important role in preventing DCM. However, requirements of amino acids in dogs are not well investigated and are presented in total dietary content basis which does not account for bioavailability or digestibility. Similarly, any nutrient (e.g., soluble and fermentable fiber) or physiological condition (e.g., size of the dog, sex, and age) that increases the requirement for taurine will also augment the possibility for DCM development. Dog food formulators should have a deep knowledge of processing methodologies and nutrient interactions beyond meeting the Association of American Feed Control Officials nutrient profiles and should not carelessly follow unsubstantiated market trends. Vegetable ingredients, including pulses, are nutritious and can be used in combination with complementary ingredients to meet the nutritional needs of the dog

Sustained release of locally delivered celecoxib provides pain relief for osteoarthritis: a proof of concept in dog patients

Objective: Drug delivery platforms that allow for gradual drug release after intra-articular administration have become of much interest as a treatment strategy for osteoarthritis (OA). The aim of this study was to investigate the safety and efficacy of an intra-articular sustained release formulation containing celecoxib (CXB), a cyclooxygenase-2 (COX-2) selective inhibitor. Methods: Amino acid-based polyesteramide microspheres (PEAMs), a biodegradable and non-toxic platform, were loaded with CXB and employed in two in vivo models of arthritis: an acute inflammatory arthritis model in rats (n = 12), and a randomized controlled study in chronic OA dog patients (n = 30). In parallel, the bioactivity of sustained release of CXB was evaluated in monolayer cultures of primary dog chondrocytes under inflammatory conditions. Results: Sustained release of CXB did not alleviate acute arthritis signs in the rat arthritis model, based on pain measurements and synovitis severity. However, in OA dog patients, sustained release of CXB improved limb function as objective parameter of pain and quality of life based on gait analysis and owner questionnaires. It also decreased pain medication dependency over a 2-month period and caused no adverse effects. Prostaglandin E2 levels, a marker for inflammation, were lower in the synovial fluid of CXB-treated dog OA patients and in CXB-treated cultured dog chondrocytes. Conclusion: These results show that local sustained release of CXB is less suitable to treat acute inflammation in arthritic joints, while safe and effective in treating pain in chronic OA in dogs

Cyclic Nucleotide Phosphodiesterases and Compartmentation in Normal and Diseased Heart

International audienceCyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cAMP and cGMP, thereby regulating multiple aspects of cardiac function. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families which are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP controlling specific cell functions in response to various neurohormonal stimuli. In myocardium, the PDE3 and PDE4 families are predominant to degrade cAMP and thereby regulate cardiac excitation-contraction coupling. PDE3 inhibitors are positive inotropes and vasodilators in human, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important to degrade cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. However, these drugs do not seem efficient in heart failure with preserved ejection fraction. There is experimental evidence that these PDEs as well as other PDE families including PDE1, PDE2 and PDE9 may play important roles in cardiac diseases such as hypertrophy and heart failure. After a brief presentation of the cyclic nucleotide pathways in cardiac cells and the major characteristics of the PDE superfamily, this chapter will present their role in cyclic nucleotide compartmentation and the current use of PDE inhibitors in cardiac diseases together with the recent research progresses that could lead to a better exploitation of the therapeutic potential of these enzymes in the future

E-Partizipation - ein notwendiges Instrument der Bürgerbeteiligung? Erfolgsfaktoren und Handlungsempfehlungen für E-Partizipationsplattformen am Beispiel MeinLB.de der Stadt Ludwigsburg

Die Arbeit stellt E-Partizipation als eine moderne und innovative Form der Bürgerbeteiligung vor und veranschaulicht am Beispiel MeinLB.de der Stadt Ludwigsburg, welche Schwierigkeiten und Herausforderungen für Beteiligungsplattformen bestehen. Zudem werden Erfolgsfaktoren und Handlungsempfehlungen für E-Partizipationsverfahren dargestellt

Subthalamic beta oscillations correlate with motor impairment in patients with Parkinson‘s disease

Einleitung: Die Tiefe Hirnstimulation (THS; Deep Brain Stimulation, DBS) ist eine seit Jahren etablierte Therapie des idiopathischen Parkinsonsyndroms (IPS), obwohl der Wirkmechanismus bisher nicht abschließend erklärt werden kann. Tiefenableitungen aus dem Nucleus subthalamicus (STN) bei IPS Patienten zeigen eine verstärkte oszillatorische Aktivität im Beta-Frequenzband (13-35 Hz), die durch dopaminerge Medikation und Tiefe Hirnstimulation (THS) unterdrückt wird, einhergehend mit einer Verbesserung der klinischen Symptome. Allerdings konnte bisher keine direkte Korrelation zwischen Beta-Ruheaktivität im OFF Zustand und den motorischen Symptomen gefunden werden. In vorherigen Studien konnte zwar indirekt ein Zusammenhang zwischen Beta-Aktivität und der Schwere des Krankheitsgrades durch relative Verbesserung der motorischen Symptome unter Medikation und parallele Abnahme der Beta-Aktivität nachgewiesen, jedoch kein direkter Zusammenhang zur OFF Symptomatik dargestellt werden. Methoden: In der hier vorliegenden Arbeit wurden bei 63 IPS-Patienten lokale Feldpotentiale (LFP) aus dem STN im medikamentösen OFF Zustand unter Ruhebedingungen bipolar von benachbarten Kontakten der THS-Elektroden abgeleitet. Powerspektren (Darstellung der Intensität der abgeleiteten Schwingungen der einzelnen Kanäle in Abhängigkeit von der Frequenz) wurden mittels Fast Fourier Transformation (FFT) für jedes Kontaktpaar als Prozent der Gesamt-Power zwischen 3–47 Hz und 53– 97 Hz errechnet. Die mittlere Power (3 Kontaktpaare pro Elektrode, beide Hemisphären) in einem breiten Beta-Band (8-35 Hz) wurde mit dem UPDRS III korreliert (bivariate zweiseitige Spearman Korrelation). Ergebnisse: Die Aktivität im Bereich von 8-35 Hz im medikamentösen OFF korreliert signifikant mit dem UPDRS III (Spearman’s ρ = 0.437, P <0.01). Bei den Untersuchungen von Subgruppen zeigte sich eine deutliche Korrelation zwischen der Aktivität von 8-35 Hz in Ruhe und dem UPDRS III der nicht- tremordominanten Gruppe (ρ = 0.417, P = 0.004, jedoch nicht für die Gruppe der tremordominanten Patienten (ρ = -0.21, P = 0.26). Diskussion: Hier zeigten wir erstmals eine direkte Korrelation der 8-35 Hz STN-Aktivität mit dem Ausmaß der motorischen Defizite bei IPS im OFF Zustand, was die potentielle Rolle der LFP-Aktivität als Biomarker für die closed-loop Stimulation, insbesondere für nicht- tremordominante Patienten, unterstützt.Introduction: Deep brain stimulation (DBS) is a well-established therapy for patients with idiopathic Parkinson’s disease (PD) although its mechanisms of action are not entirely understood yet. In PD patients the commonly used target for DBS is the subthalamic nucleus (STN). DBS gives the unique opportunity to record neuronal activity from the target structures for stimulation. Over the last decade several studies have shown enhanced oscillatory activity in the beta band (13-35Hz) recorded from the STN in PD patients. This beta activity is suppressed by dopaminergic medication and DBS, accompanied by the improvement of the clinical symptoms. So far, no direct correlation of the beta activity at rest after withdrawal of dopaminergic medication and the motor symptoms of PD patients have been found. Earlier studies showed correlations between relative improvement in motor symptoms and modulation of STN beta activity with medication. Methods: This study explores the local field potential (LFP) activity recorded from the STN in 63 PD patients undergoing DBS. All recordings were obtained at least 12h after withdrawal of dopaminergic medication (OFF medication). The power spectra were analyzed as a percentage of the total power over a frequency range of 3-47 Hz and 53- 97 Hz by means of a Fast Fournier Transformation (FFT). The power-spectra of a broad beta frequency band (8-35 Hz) were correlated with the Unified Parkinson’s Disease Rating Scale Part III (UPDRS-III) in the OFF medication state. Further, subgroup analysis was performed for tremor-dominant patients and akinetic-rigid patients. Results: The activity in the range of 8-35 Hz and the total UPDRS-III at the OFFmedication state correlated significantly across all patients (Spearman’s ρ = 0.437, P< 0.01). Within the subgroups we showed a significant correlation between the 8-35Hz band power and the UPDRS III just within the group of bradykinetic-rigid patients (ρ = 0.417, P= 0.004), not for the tremor dominant group (ρ = -0.21, P = 0.26). Discussion: Our results showed a direct correlation between the local STN power between 8-35Hz and the motor impairment in bradykinetic-rigid PD patients. These results support the role of the subthalamic oscillatory activity as a potential biomarker for closed loop stimulation for Parkinson’s disease

A new innovative real-time tracking method for flying insects applicable under natural conditions

Background Sixty percent of all species are insects, yet despite global efforts to monitor animal movement patterns, insects are continuously underrepresented. This striking difference between species richness and the number of species monitored is not due to a lack of interest but rather to the lack of technical solutions. Often the accuracy and speed of established tracking methods is not high enough to record behavior and react to it experimentally in real-time, which applies in particular to small flying animals. Results Our new method of real-time tracking relates to frequencies of solar radiation which are almost completely absorbed by traveling through the atmosphere. For tracking, photoluminescent tags with a peak emission (1400 nm), which lays in such a region of strong absorption through the atmosphere, were attached to the animals. The photoluminescent properties of passivated lead sulphide quantum dots were responsible for the emission of light by the tags and provide a superb signal-to noise ratio. We developed prototype markers with a weight of 12.5 mg and a diameter of 5 mm. Furthermore, we developed a short wave infrared detection system which can record and determine the position of an animal in a heterogeneous environment with a delay smaller than 10 ms. With this method we were able to track tagged bumblebees as well as hawk moths in a flight arena that was placed outside on a natural meadow. Conclusion Our new method eliminates the necessity of a constant or predictable environment for many experimental setups. Furthermore, we postulate that the developed matrix-detector mounted to a multicopter will enable tracking of small flying insects, over medium range distances (>1000m) in the near future because: a) the matrix-detector equipped with an 70 mm interchangeable lens weighs less than 380 g, b) it evaluates the position of an animal in real-time and c) it can directly control and communicate with electronic devices