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Biology subjects

Claus, P.

Publications and source records attributed to Claus, P..

10 recordsLinked to original sources

Directed evolution of the Fe-nitrogenase for CO2 reduction to hydrocarbons

The iron (Fe) nitrogenase drives bacterial methane (CH4) formation by converting carbon dioxide (CO2) to CH4 in a single enzymatic step. Enhancing the initial CH4 formation activity of Fe-nitrogenase and expanding the product spectrum to hydrocarbon chains could lead to a route for sustainable feedstock chemicals. Here, we performed the first directed evolution campaign on the Fe-nitrogenase aimed at optimizing the hydrocarbon production. We achieved an [~]8-fold increase in CH4 formation by Fe-nitrogenase expressing Rhodobacter capsulatus cultures in three rounds of site-saturation mutagenesis. The best performing mutant (F362ManfD, Y85FanfD, T360SanfD) extends the in vivo product spectrum of the nitrogenase to ethane (C2H6) and exhibits 6-fold higher rates for CO production in vitro, whereas the formation of the undesirable byproduct formate was abolished. Electron microscopy-based structural analysis identified a methionine and water potentially stabilizing the transition state and fine-tuning the CO2 reduction mechanism and activity.

biochemistry↗

Form IF Rubiscos include highly active, specific, and small subunit-independent enzymes.

Plant-type (Form I) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) suffers from inherent catalytic trade-offs and a strong dependency on other proteins--including an essential small subunit (SSU) and auxiliary chaperones--for assembly, constraining the enzymes evolutionary and engineering potential. Here, we investigated representatives from the newly discovered clade Form IF. These enzymes do not require specific chaperones to form functional complexes, exhibit high CO2-specificities (SC/O [~]50) while maintaining high turnover rates (up to kcat [~]11 s-1). Remarkably, two Form IF representatives (IF-1/IF-2) lost the dependency on the SSU and assemble into homo-octameric complexes without their cognate SSUs. While the SSU is not necessary for catalysis, its addition improves both activity and specificity in IF-1/IF-2. Our results show that complexity is actually not required to achieve highly active, specific and functional Rubisco variants--and that this complexity can even be reverted--which challenges our current thinking on the evolution and catalytic mechanism of Rubisco.

biochemistry↗

Combining evolution and machine learning-guided pathway optimization to engineer a novel methylsuccinate module for synthetic C1 metabolism in vivo

De novo metabolic pathways open possibilities for sustainable biotransformations in microbes. However, the in vivo-implementation of such new-to-nature pathways is highly challenging and heavily relies on adaptive laboratory evolution (ALE) of the hosts native metabolic network. Here, we assess how much this need for host-centric ALE can be overcome and/or complemented through the informed design of the newly introduced pathway. Exemplifying for a synthetic CO2-fixation module via methyl-succinate, we established methylsuccinate-dependent growth of Escherichia coli over six months by ALE of E. colis native metabolism. In parallel, we developed a machine-learning guided workflow (MEVIS) for the automated engineering of the synthetic pathway, resulting in methylsuccinate-dependent growth within three weeks. Critically, performing MEVIS in the background of the ALE-evolved strain is necessary to further approach wild-type like growth, demonstrating how ALE in combination with machine-learning-guided lab automation holds great potential to accelerate and improve design-build-test-learn cycles in contemporary metabolic engineering.

bioengineering↗

Integrated multi-modal data analysis for computational modeling of healthy and location-dependent myocardial infarction conditions in porcine hearts

Porcine hearts are widely used for preclinical cardiac evaluation. Computational models, by effectively integrating comprehensive experimental data, often reinforce this preclinical assessment. Using extensive multi-modal data, we developed swine ventricular digital twins for healthy and chronic myocardial infarction (MI) conditions to investigate the roles of the cardiac conduction system (CS), spatial repolarization heterogeneities, cardiomyocyte orientation, cell-to-cell coupling, and MI characteristics on ventricular function. We analyzed cardiac magnetic resonance (CMR) images, electrocardiograms (ECGs), and optical (OM) and electroanatomical mapping from 5 healthy and 10 MI pigs. CS architectures were built from OM and ECG recordings. Myocardial fiber orientation, action potential characteristics, and cell-to-cell conductivity in MI tissue were defined from OM and CMR data. Simulated ECGs for healthy and MI models of left anterior descending and left circumflex occlusions were compared to experimental ECGs and used to assess MI-induced changes. Personalized fiber orientation minimally affected electrophysiology, with conduction velocity (CV) and action potential duration (APD) changing less than 3.6% with respect to standard orientation. Accurate CS and repolarization heterogeneities reproduced depolarization (0.76 QRS similarity) and repolarization (0.74 T-wave similarity) patterns. Incorporating experimentally guided MI-induced alterations enabled replication of MI depolarization and repolarization features (relative errors: 0.5% CV, 2.9% APD), yielded realistic T-wave morphologies (0.63 similarity), and revealed ECG patterns specific to vessel-dependent occlusions. Thus, by integrating extensive multi-modal data, we advance porcine cardiac digital twins and demonstrate the influence of key structural and electrophysiological parameters on healthy and MI heart function, providing a robust computational framework for mechanistic and translational applications. Author summaryPigs are commonly used as preclinical models for cardiac evaluation due to their close resemblance to the human heart. Computational cardiac electrophysiology often supports and extends this preclinical assessment. However, the reliability of these in silico representations depends on the effective integration of comprehensive experimental data. Using extensive multi-modal data, we developed swine ventricular digital twins under healthy and chronic myocardial infarction conditions. These models allowed us to investigate the influence of the cardiac conduction system, spatial repolarization heterogeneities, cardiomyocyte orientation, cell-to-cell coupling, and vessel-specific infarction characteristics on ventricular function. Our results show that cardiomyocyte orientation exerts only a minor effect on electrophysiology, with average conduction velocity showing a slight decrease and action potential duration remaining unchanged when comparing standard versus personalized orientations. Accurate representation of conduction system architecture and repolarization heterogeneities enabled closed reproduction of experimental depolarization and repolarization patterns. Furthermore, by integrating experimentally guided reductions in cell-to-cell coupling and inward rectifier potassium current, along with personalized post-infarction activation alterations and a novel porcine cellular model, we were able to faithfully replicate infarction-specific depolarization-repolarization features. These refinements produced realistic T-wave morphologies and revealed electrocardiographic signatures associated with vessel-dependent infarctions, underscoring the translational potential of our approach.

bioengineering↗

Translation-specific disruption of Col1a1 expression in multiple models of Spinal Muscular Atrophy can be rescued by Risdiplam.

Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disorder caused by decreased levels of Survival of Motor Neuron (SMN) protein. If left untreated, SMA patients have a poor prognosis, marked by the degeneration of motor neurons, progressive muscle weakness and atrophy. The approval of SMN-restoring therapies that improve symptoms and lifespan in patients with SMA has created emerging, non-neuronal phenotypes and an urgent need for deepening our understanding of disease pathogenesis. Leveraging the knowledge that SMN loss drives alterations in translation, we used multiple tissues from a mouse model of SMA to uncover early translational alterations in key mRNAs and proteins, which act as contributors to pathogenesis and hallmarks of the disease. Among hundreds of differentially translated mRNAs, Col1a1 emerged as a translation-specific manifestation of early defects in the mouse model. These findings were confirmed in fibroblasts derived from patients with varying levels of disease severity. Notably, treatment with SMN-restoring therapies rescued COL1A1 protein levels, particularly in fibroblasts from patients with the most severe forms of the disease. Overall, our study identifies COL1A1 as an indicator of disease severity in SMA, which captures early molecular alterations and respond to SMN-modifying therapies.

molecular biology↗

Bottom-up reconstruction of minimal pyrenoids provides insights into the evolution and mechanisms of carbon concentration by EPYC1 proteins

Membraneless organelles play essential roles in cellular processes. In various photosynthetic organisms, they confer carbon concentrating mechanisms (CCMs). One example is the pyrenoid in Chlamydomonas reinhardtii, a liquid-phase separated organelle that localizes and improves carbon fixation via the intrinsically-disordered protein (IDP) EPYC1. Modern-day pyrenoids are complex structures, which makes it impossible to study the function of EPYC1, especially whether EPYC1 alone confers carbon concentration, and how EPYC1 could have initiated the evolution of pyrenoids. Here, we developed a bottom-up approach to study the function of EPYC1 and its sequence-function space across evolution. We demonstrate that modern-day EPYC1-sequences, but not other IDPs, induce liquid-phase separation of Rubisco into minimal pyrenoids with functional CCMs. Using ancestral sequence reconstruction, we trace the evolution of pyrenoids and demonstrate that selection acted on carboxylation rate, and in selected cases on specificity, providing unexpected perspectives on the design and function of natural and synthetic pyrenoids.

biochemistry↗

Precision sampling of discrete sites identified during in-vivo functional testing in the mammalian heart

Ventricular arrhythmias after myocardial infarction (MI) originate from discrete areas within the MI border zone (BZ), identified during functional electrophysiology tests. Accurate sampling of arrhythmogenic sites for ex vivo study remains challenging, yet is critical to identify their tissue, cellular and molecular signature. In this study, we developed, validated, and applied a targeted sampling methodology based on individualized 3D prints of the human-sized pig heart. To this end, 3D anatomical models of the left ventricle were created from magnetic resonance imaging and fused with biplane fluoroscopy. Regions of interest for sampling were annotated on the anatomical models, from which we created a unique 3D printed cast with custom slits identifying the annotated regions for sampling. The methodology was validated by retrieving ablation lesions created at predefined locations on the anatomical model. We applied the methodology to sample arrhythmia-vulnerable regions after MI during adrenergic stimulation. A novel pipeline of imaging was developed to create a 3D high-resolution map of each sample, highlighting the complex interplay of cellular organization, and altered innervation in the BZ.

bioengineering↗

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolones activities. Using an RNA-sequencing, bioinformatics and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone- treated and untreated Smn-/-;SMN2 SMA and Smn+/-;SMN2 healthy mice, we identified molecular targets linked to prednisolones ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolones potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

pathology↗

Dysregulation of the Tweak/Fn14 pathway in skeletal muscle of spinal muscular atrophy mice

Spinal muscular atrophy (SMA) is a childhood neuromuscular disorder caused by depletion of the survival motor neuron (SMN) protein. SMA is characterized by the selective death of spinal cord motor neurons, leading to progressive muscle wasting. Loss of skeletal muscle in SMA is a combination of denervation-induced muscle atrophy and intrinsic muscle pathologies. Elucidation of the pathways involved is essential to identify the key molecules that contribute to and sustain muscle pathology. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/TNF receptor superfamily member fibroblast growth factor inducible 14 (Fn14) pathway has been shown to play a critical role in the regulation of denervation-induced muscle atrophy as well as muscle proliferation, differentiation and metabolism in adults. However, it is not clear whether this pathway would be important in highly dynamic and developing muscle. We thus investigated the potential role of the TWEAK/Fn14 pathway in SMA muscle pathology, using the severe Taiwanese Smn-/-;SMN2 and the less severe Smn2B/- SMA mice, which undergo a progressive neuromuscular decline in the first three post-natal weeks. Here, we report significantly dysregulated expression of the TWEAK/Fn14 pathway during disease progression in skeletal muscle of the two SMA mouse models. In addition, siRNA-mediated Smn knockdown in C2C12 myoblasts suggests a genetic interaction between Smn and the TWEAK/Fn14 pathway. Further analyses of SMA, Tweak-/- and Fn14-/- mice revealed dysregulated myopathy, myogenesis and glucose metabolism pathways as a common skeletal muscle feature, and providing further evidence in support of a relationship between the TWEAK/Fn14 pathway and Smn. Finally, a pharmacological intervention (Fc-TWEAK) to upregulate the activity of the TWEAK/Fn14 pathway improved disease phenotypes in the two SMA mouse models. Our study provides novel mechanistic insights into the molecular players that contribute to muscle pathology in SMA and into the role of the TWEAK/Fn14 pathway in developing muscle.

pathology↗

Muscle overexpression of Klf15 via an AAV8-Spc5-12 construct does not provide benefits in spinal muscular atrophy mice

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the survival motor neuron (SMN) gene. While there are currently two approved gene-based therapies for SMA, availability, high cost, and differences in patient response indicate that alternative treatment options are needed. Optimal therapeutic strategies will likely be a combination of SMN-dependent and -independent treatments aimed at alleviating symptoms in the central nervous system and peripheral muscles. Kruppel-like factor 15 (KLF15) is a transcription factor that regulates key metabolic and ergogenic pathways in muscle. We have recently reported significant downregulation of Klf15 in muscle of pre-symptomatic SMA mice. Importantly, perinatal upregulation of Klf15 via transgenic and pharmacological methods resulted in improved disease phenotypes in SMA mice, including weight and survival. In the current study, we designed an adeno-associated virus serotype 8 (AAV8) vector to overexpress a codon-optimised Klf15 cDNA under the muscle-specific Spc5-12 promoter (AAV8-Klf15). Administration of AAV8-Klf15 to severe Taiwanese Smn-/-;SMN2 or intermediate Smn2B/- SMA mice significantly increased Klf15 expression in muscle. We also observed significant activity of the AAV8-Klf15 vector in liver and heart. AAV8-mediated Klf15 overexpression moderately improved survival in the Smn2B/- model but not in the Taiwanese mice. An inability to specifically induce Klf15 expression at physiological levels in a time- and tissue-dependent manner may have contributed to this limited efficacy. Thus, our work demonstrates that an AAV8-Spc5-12 vector induces high gene expression as early as P2 in several tissues including muscle, heart and liver, but highlights the challenges of achieving meaningful vector-mediated transgene expression of Klf15.

pathology↗