Network design and analysis for multi-enzyme biocatalysis

In vitro synthesis is a biotechnological alternative to classic chemical catalysts. However, the manual design of multi-step biosynthesis routes is very challenging, especially when enzymes from different organisms are involved. There is therefore a demand for in silico tools to guide the design of such synthesis routes using computational methods for the path-finding, as well as the reconstruction of suitable genome-scale metabolic networks that are able to harness the growing amount of biological data available. This work presents an algorithm for finding pathways from arbitrary metabolites to a target product of interest. The algorithm is based on a mixed-integer linear program (MILP) and combines graph topology and reaction stoichiometry. The pathway candidates are ranked using different ranking criteria to help finding the best suited synthesis pathway candidates. Additionally, a comprehensive workflow for the reconstruction of metabolic networks based on data of the Kyoto Encyclopedia of Genes and Genomes (KEGG) combined with thermodynamic data for the determination of reaction directions is presented. The workflow comprises a filtering scheme to remove unsuitable data. With this workflow, a panorganism network reconstruction as well as single organism network models are established. These models are analyzed with graph-theoretical methods. It is also discussed how the results can be used for the planning of biosynthetic production pathways.In vitro Synthese ist eine biotechnologische Alternative zu klassischen chemischen Katalysen. Der manuelle Entwurf von mehrstufigen Biosynthesewegen ist jedoch sehr anspruchsvoll, vor allem wenn Enzyme verschiedener Organismen beteiligt sind. Daher besteht ein Bedarf an Methoden, die helfen solche Synthesewege in silico zu entwerfen und die in der Lage sind große Mengen biologischer Daten zu bewältigen - insbesondere in Hinblick auf die Rekonstruktion genomskaliger metabolischer Netzwerkmodelle und die Pfadsuche in solchen Netzwerken. In dieser Arbeit wird ein Algorithmus zur Pfadsuche zu einem Zielprodukt ausgehend von beliebigen Substraten präsentiert. Der Algorithmus basiert auf einem gemischt-ganzzahligen linearen Programm, das Graphtopologie mit Reaktionsstöchiometrien kombiniert. Die Pfadkandidaten werden anhand verschiedener Kriterien geordnet, um die am besten geeigneten Kandidaten für die Synthese zu finden. Außerdem wird ein umfassender Workflow für die Rekonstruktion metabolischer Netzwerke basierend auf der Datenbank KEGG sowie thermodynamischen Daten vorgestellt. Dieser umfasst einen Filter, der anhand verschiedener Kriterien geeignete Reaktionen auswählt. Der Workflow wird zum Erstellen einer organismusübergreifenden Netzwerkrekonstruktion, sowie Netzwerken einzelner Organismen genutzt. Diese Modelle werden mit graphentheoretischen Methoden analysiert. Es wird diskutiert, wie die Ergebnisse für die Planung von biosynthetischen Produktionswegen genutzt werden können.BMBF; Initiative “Biotechnologie 2020+: Basistechnologien für eine nächste Generation biotechnologischer Verfahren”; Projekt MECA

In-depth characterization of genome-scale network reconstructions for the in vitro synthesis in cell-free systems

Cell‐free systems containing multiple enzymes are becoming an increasingly interesting tool for one‐pot syntheses of biochemical compounds. To extensively explore the enormous wealth of enzymes in the biological space, we present methods for assembling and curing data from databases to apply them for the prediction of pathway candidates for directed enzymatic synthesis. We use Kyoto Encyclopedia of Genes and Genomes to establish single organism models and a pan‐organism model that is combining the available data from all organisms listed there. We introduce a filtering scheme to remove data that are not suitable, for example, generic metabolites and general reactions. In addition, a valid stoichiometry of reactions is required for acceptance. The networks created are analyzed by graph theoretical methods to identify a set of metabolites that are potentially reachable from a defined set of starting metabolites. Thus, metabolites not connected to such starting metabolites cannot be produced unless new starting metabolites or reactions are introduced. The network models also comprise stoichiometric and thermodynamic data that allow the definition of constraints to identify potential pathways. The resulting data can be directly applied using existing or future pathway finding tools

Ablation of C-type natriuretic peptide/cGMP signaling in fibroblasts exacerbates adverse cardiac remodeling in mice

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counter-regulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerted antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor.CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of AngiotensinII (AngII) and TGF-β. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to AngII infusions. Moreover, after two weeks of mild pressure-overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes collagen I, III and periostin. Notably, such responses to AngII and TAC were greater in female as compared to male KO mice. Enhanced AngII-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity.The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling

Heart-Microcirculation Connection

Cardiac ANP (atrial natriuretic peptide) moderates arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve inhibition of the renin-angiotensin-aldosterone system, increased natriuresis, endothelial permeability, and vasodilatation. The contribution of the direct vasodilating effects of ANP to blood pressure homeostasis is controversial because variable levels of the ANP receptor, GC-A (guanylyl cyclase-A), are expressed among vascular beds. Here, we show that ANP stimulates GC-A/cyclic GMP signaling in cultured microvascular pericytes and thereby the phosphorylation of the regulatory subunit of myosin phosphatase 1 by cGMP-dependent protein kinase I. Moreover, ANP prevents the calcium and contractile responses of pericytes to endothelin-1 as well as microvascular constrictions. In mice with conditional inactivation (knock-out) of GC-A in microcirculatory pericytes, such vasodilating effects of ANP on precapillary arterioles and capillaries were fully abolished. Concordantly, these mice have increased blood pressure despite preserved renal excretory function. Furthermore, acute intravascular volume expansion, which caused release of cardiac ANP, did not affect blood pressure of control mice but provoked hypertensive reactions in pericyte GC-A knock-out littermates. We conclude that GC-A/cGMP-dependent modulation of pericytes and microcirculatory tone contributes to the acute and chronic moderation of arterial blood pressure by ANP. Graphic Abstract Ais available for this article

La relazione degli amministratori

Il bilancio d'esercizio si compone di una parte tecnico-contabile e di una parte descrittivo-integrativa, formata da numerosi documenti che, nel complesso, costituiscono il corredo integrativo al bilancio tecnico-contabile. Tra i numerosi documenti, la relazione sulla gestione, la nota integrativa e il rendiconto finanziario sono i più significativi in quando comprendono dati senza i quali sarebbe impossibile leggere e interpretare i dati di Stato Patrimoniale e di Conto Economico. Questo studio esamina la funzione e il contenuto di quelle tre fondamentali parti del bilancio, indicando i criteri per la loro corretta redazione

Measurement of the top quark pole mass using tt‾ \textrm{t}\overline{\textrm{t}} +jet events in the dilepton final state in proton-proton collisions at s \sqrt{s} = 13 TeV

A measurement of the top quark pole mass ${{m_{\mathrm{t}}} ^{\text{pole}}}$ in events where a top quark-antiquark pair ($\mathrm{t\bar{t}}$) is produced in association with at least one additional jet ($\mathrm{t\bar{t}}$+jet) is presented. This analysis is performed using proton-proton collision data at $\sqrt{s} =$ 13 TeV collected by the CMS experiment at the CERN LHC, corresponding to a total integrated luminosity of 36.3 fb$^{-1}$. Events with two opposite-sign leptons in the final state (e$^{+}$e$^{-}$, $\mu^{+}\mu^{-}$, e$^{\pm}\mu^{\mp}$) are analyzed. The reconstruction of the main observable and the event classification are optimized using multivariate analysis techniques based on machine learning. The production cross section is measured as a function of the inverse of the invariant mass of the $\mathrm{t\bar{t}}$+jet system at the parton level using a maximum likelihood unfolding. Given a reference parton distribution function (PDF), the top quark pole mass is extracted using the theoretical predictions at next-to-leading order. For the ABMP16NLO PDF, this results in ${{m_{\mathrm{t}}} ^{\text{pole}}} =$ 172.94 $\pm$ 1.37 GeV.A measurement of the top quark pole mass ${m}_{\textrm{t}}^{\textrm{pole}}$ in events where a top quark-antiquark pair ($\textrm{t}\overline{\textrm{t}}$) is produced in association with at least one additional jet ($\textrm{t}\overline{\textrm{t}}$+jet) is presented. This analysis is performed using proton-proton collision data at $\sqrt{s}$ = 13 TeV collected by the CMS experiment at the CERN LHC, corresponding to a total integrated luminosity of 36.3 fb$^{−1}$. Events with two opposite-sign leptons in the final state (e$^{+}$e$^{−}$, μ$^{+}$μ$^{−}$, e$^{±}$μ$^{∓}$) are analyzed. The reconstruction of the main observable and the event classification are optimized using multivariate analysis techniques based on machine learning. The production cross section is measured as a function of the inverse of the invariant mass of the $\textrm{t}\overline{\textrm{t}}$+jet system at the parton level using a maximum likelihood unfolding. Given a reference parton distribution function (PDF), the top quark pole mass is extracted using the theoretical predictions at next-to-leading order. For the ABMP16NLO PDF, this results in ${m}_{\textrm{t}}^{\textrm{pole}}$ = 172.93 ± 1.36 GeV.[graphic not available: see fulltext]A measurement of the top quark pole mass $m_\mathrm{t}^\text{pole}$ in events where a top quark-antiquark pair ($\mathrm{t\bar{t}}$) is produced in association with at least one additional jet ($\mathrm{t\bar{t}}$+jet) is presented. This analysis is performed using proton-proton collision data at $\sqrt{s}$ = 13 TeV collected by the CMS experiment at the CERN LHC, corresponding to a total integrated luminosity of 36.3 fb$^{-1}$. Events with two opposite-sign leptons in the final state (e$^+$e$^-$, $\mu^+\mu^-$, e$^\pm\mu^\mp$) are analyzed. The reconstruction of the main observable and the event classification are optimized using multivariate analysis techniques based on machine learning. The production cross section is measured as a function of the inverse of the invariant mass of the $\mathrm{t\bar{t}}$+jet system at the parton level using a maximum likelihood unfolding. Given a reference parton distribution function (PDF), the top quark pole mass is extracted using the theoretical predictions at next-to-leading order. For the ABMP16NLO PDF, this results in $m_\mathrm{t}^\text{pole}$ = 172.93 $\pm$ 1.36 GeV

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field