Biology subjects

Harms, H.

Publications and source records attributed to Harms, H..

9 recordsLinked to original sources

bioRxiv2025

Framework for combined functional and computational assessment of variant pathogenicity in the sodium leak channel NALCN

The sodium leak channel nonselective (NALCN) is the pore-forming channel component of the NALCN channelosome. This protein complex generates a depolarizing sodium leak in various tissues and contributes to respiratory and circadian rhythms, locomotion, and sleep. De novo and inherited variants of NALCN can lead to severe developmental syndromes called contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD) and infantile hypotonia with psychomotor retardation and characteristic facies 1 (IHPRF1), respectively. Although variants of uncertain significance (VUS) or presumed pathogenic variants have been studied in heterologous expression systems before, there is no generally accepted framework on how to assess or predict variant pathogenicity. We set out use the functional and computational characterization of 19 VUS detected in CLIFAHDD and IHPRF1 patients to establish a robust analysis to classify suspected disease-causing variants. Specifically, we employ a combination of multiple parameters derived from two-electrode voltage-clamp electrophysiology recordings and predicted protein stability and conservation scores. We show that this approach is capable of distinguishing benign common variants from both gain- and loss-of-function (GoF/LoF) variants. Additionally, our work provides mechanistic insight into the molecular mechanism underlying specific variants and provides insight into the unusual propensity of NALCN missense variants to result in GoF phenotypes. We anticipate that this experimental and computational framework will aid assessment of variant pathogenicity of NALCN and other components of the channelosome in the future.

genetics↗

bioRxiv2025

A double-staining automated flow cytometry method for real-time monitoring of bacteria in continuous bioreactors

In industrial biotechnology, cell density is a critical parameter that influences key process control variables such as feed rate, harvest timing, and product recovery. Traditional biomass measurements are indirect, incapable of quantifying population heterogeneity, and often prone to inaccuracies due to manual handling and interference from culture media compounds. This study introduces an automated flow cytometry approach to enable continuous, real-time monitoring of bacterial cultures in a continuous bioreactor. Our method employs a double-staining protocol that combines DAPI for assessing total DNA content with Alexa Fluor 488 via Click-iT technology to detect the percentage of cells with active DNA replication through EdU incorporation. This streamlined protocol, which integrates cell fixation, permeabilization, staining, and measurement, was applied to three Gram-negative strains: Bradyrhizobium sp., Escherichia coli, and Stenotrophomonas rhizophila. The approach successfully captured both growth dynamics and cell cycle progression, providing rapid and quantitative insights into culture composition. The enhanced temporal resolution achieved by this method facilitates timely adjustments of process parameters, ultimately improving bioprocess efficiency and product quality. These results underscore the potential of automated flow cytometry as a powerful tool for real-time monitoring and control in industrial biotechnology.

microbiology↗

bioRxiv2022

Functional redundancy secures resilience of chain elongation communities upon pH shifts in closed bioreactor ecosystems

For anaerobic mixed cultures performing microbial chain elongation, it is unclear how pH alterations affect the abundance of key players, microbial interactions and community functioning in terms of medium-chain carboxylate yields. We explored pH effects on mixed cultures enriched in continuous anaerobic bioreactors representing closed model ecosystems. Increasing the pH from 5.5 to 6.0 caused fluctuations in community composition and yields of n-butyrate, n-caproate and n-caprylate. Further pH increase to 6.5 restored previous yield values while the community entered a new state characterized by lower diversity and evenness but apparently higher richness, indicating the presence of species below the detection threshold in the previous state. We applied Aitchison PCA clustering, linear mixed-effects models and random forest classification on our datasets. Different pH preferences of two key chain elongation species - one Clostridium IV species related to Ruminococcaceae bacterium CPB6, and one Clostridium sensu stricto species related to Clostridium luticellarii - were determined. Based on relative abundances measured by 16S rRNA amplicon sequencing, network analysis revealed positive correlations of Clostridium IV with lactic acid bacteria, which switched from Olsenella to Lactobacillus along the pH increase, illustrating the plasticity of the food web in chain elongation communities. The pH increase induced dramatic shifts in community composition whereas process performance (in terms of product range and yields) returned to the previous state. Despite long-term cultivation in closed systems over the pH shift experiment, the communities retained functional redundancy in fermentation pathways, reflected by the emergence of rare species and concomitant recovery of chain elongation functions.

microbiology↗

bioRxiv2022

Impact of fungal hyphae on growth and dispersal of obligate anaerobic bacteria in aerated habitats

Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes even in well-areated environments. Here, we analyzed whether and to which degree fractal hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches allowing for spore germination, growth and fermentative activity of the obligate anaerobe in an otherwise oxic environment. Hypoxic liquid films containing 80 {+/-} 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Our results suggest that fungal biomass typical in soil (<550 g g-1soil) may create anoxic microniches and enable activity as well as dispersal of obligate anaerobes near hyphae in an otherwise inhabitable environment.

microbiology↗

bioRxiv2021

Phage co-transport with hyphal-riding bacteria fuels bacterial invasion in water-unsaturated microbial ecosystems

Non-motile microbes enter new habitats often by co-transport with motile microorganisms. Here, we report on the ability of hyphal-riding bacteria to co-transport lytic phages and utilize them as weapons during colonization of new water-unsaturated habitats. This is comparable to the concept of biological invasions in macroecology. In analogy to invasion frameworks in plant and animal ecology, we tailored spatially organized, water-unsaturated model microcosms using hyphae of Pythium ultimum as invasion paths and flagellated soil-bacterium Pseudomonas putida KT2440 as carrier for co-transport of Escherichia virus T4. P. putida KT2440 efficiently dispersed along P. ultimum to new habitats and dispatched T4 phages across air gaps transporting {approx} 0.6 phages bacteria-1. No T4 displacement along hyphae was observed in the absence of carrier bacteria. If E. coli occupied the new habitat, T4 co-transport fueled the fitness of invading P. putida KT2440, while the absence of phage co-transport led to poor colonization followed by extinction. Our data emphasize the importance of hyphal transport of bacteria and associated phages in regulating fitness and composition of microbial populations in water-unsaturated systems. As such co-transport mirrors macroecological invasion processes, we recommend hyphosphere systems with motile bacteria and co-transported phages as models for testing hypotheses in invasion ecology.

microbiology↗

bioRxiv2020

Functional crosstalk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive post-translational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable post-translational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semi-synthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both WT and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity towards the clinically used anti-arrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens. Significance statementThe cardiac sodium channel (Nav1.5) is crucial for generating a regular heartbeat. It is thus not surprising that mutations in its sequence have been linked to life-threatening arrhythmias. Interestingly, Nav1.5 activity can also be altered by posttranslational modifications, such as tyrosine phosphorylation. Our combination of protein engineering and molecular modeling studies has revealed that the detrimental effect of a long QT3 patient mutation is only exposed when a proximal tyrosine is phosphorylated. This suggests a dynamic crosstalk between the genetic mutation and a neighboring phosphorylation, a phenomenon that could be important in other classes of proteins. Additionally, we show that phosphorylation can affect the channels sensitivity towards clinically-relevant drugs, a finding that may prove important when devising patient-specific treatment plans.

biophysics↗

bioRxiv2020

Microbial resource management for ex situ biomethanation of hydrogen at alkaline pH

Biomethanation is a promising solution to convert H2 produced from surplus electricity and CO2 to CH4 by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H2/CO2. We enriched a hydrogenotrophic microbiota on CO2 and H2 as sole carbon and energy sources, respectively, to investigate these competing reactions. Microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched as acetate was concomitantly produced along with CH4. By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH4 ([≥] 97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities [≥]1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus. The bacterial community was dominated by Lutispora. The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards selective production of CH4 from H2/CO2 by adapting the media composition, the reducing agent and the stirring intensity.

bioengineering↗

bioRxiv2020

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Manipulation of proteins by chemical modification is a powerful way to decipher their function or harness that function for therapeutic purposes. Despite recent progress in ribosome-dependent and semi-synthetic chemical modifications, these techniques sometimes have limitations in the number and type of modifications that can be simultaneously introduced or their application in live eukaryotic cells. Here we present a new approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into soluble and membrane proteins expressed in eukaryotic cells. We insert synthetic peptides into proteins of interest via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating different aspects of GFP, NaV1.5 and P2X2 receptor function. Because the approach can introduce virtually any chemical modification into both intracellular and extracellular regions of target proteins, we anticipate that it will overcome some of the drawbacks of other semi-synthetic or ribosome-dependent methods to engineer proteins.

synthetic biology↗

bioRxiv2019

Resilience trinity: safeguarding ecosystem services across three different time horizons and decision contexts

Ensuring ecosystem resilience is an intuitive approach to safeguard future provisioning of ecosystem services (ES). However, resilience is an ambiguous concept and difficult to operationalize. Focusing on resilience mechanisms, such as diversity, network architectures or adaptive capacity, has recently been suggested as means to operationalize resilience. Still, the focus on mechanisms is not specific enough because the usefulness of a mechanism is context-dependent. We suggest a conceptual framework, resilience trinity, to facilitate management of resilience mechanisms in three distinctive decision contexts and time-horizons. i) reactive, when there is an imminent threat to ES resilience and a high pressure to act, ii) adjustive, when the threat is known in general but there is still time to adapt management, and iii) provident when time horizons are very long and the nature of the threats is uncertain, leading to a low willingness to act. This emphasizes that resilience has different interpretations and implications at different time horizons which however need to be reconciled. The inclusion of time into resilience thinking ensures that longer-term management actions are not missed while urgent threats to ES are given priority.

ecology↗