Biology subjects

Kern, J.

Publications and source records attributed to Kern, J..

9 recordsLinked to original sources

bioRxiv2026

Glomerular Endothelial Cell-Derived Extracellular Vesicles Cross the Basement Membrane to Regulate Podocyte Function

BackgroundSmall extracellular vesicles (EVs) are nanosized, endosome-derived particles which transfer RNA, proteins, and bioactive molecules to mediate intercellular communication. While EV signaling has been observed in many organ systems, it remains unclear whether glomerular endothelial cell (GEC)-derived small EVs directly interact with podocytes in vivo or how they traverse the glomerular basement membrane (GBM). MethodsGEC-derived small EVs were characterized by nanoparticle tracking analysis, electron microscopy, RAMAN spectroscopy and flow cytometry. Cargo composition was analyzed by proteomics, and microRNA (miR) profiling. Functional and structural features were examined using protease, collagenase, adhesion, and multimodal imaging assays. GEC-derived small EV uptake and downstream transcriptional effects were studied in cultured podocytes, while in vivo trafficking was assessed by injection of labeled small EVs into transgenic zebrafish larvae under baseline conditions, puromycin-induced damage, and cd2ap-knockdown. ResultsGECs released bona fide exosome-like small EVs carrying a highly cell type-specific miR cargo. Small EV transfer to podocytes induced a defined transcriptional response consistent with miR-mediated repression of target genes involved in extracellular matrix organization, cell cycle regulation, and cellular stress responses. Proteomic analyses revealed enrichment of surface proteases and integrin-associated proteins that conferred sustained proteolytic activity and enabled GEC-derived small EV migration through extracellular matrix surrogates. In vivo, circulating small EVs traversed the GBM and localized selectively to podocytes in healthy glomeruli, whereas glomerular injury permitted small EV entry into the tubular compartment. ConclusionThese findings provide first in vivo evidence that GEC-derived small EVs can cross the GBM and impact on podocytes. By identifying integrin- and protease-dependent mechanisms which facilitate vesicle passage, this study redefines the GBM as a dynamic interface of heterocellular, vesicle-mediated communication.

physiology↗

bioRxiv2025

Atomic resolution structure of spinach rubisco reveals protons and dynamics

Photosynthetic organisms sustain life on Earth by storing solar energy in biomass. Central to this process is rubisco, the enzyme that catalyses the fixation of CO2 to ribulose-1,5-bisphosphate, providing the primary gateway for inorganic carbon into the biosphere. Rubiscos catalytic efficiency is a major determinant of crop productivity and global carbon flux, making it a longstanding target for protein engineering.1-4 Yet, attempts to enhance its performance through rational design have met limited success due to an incomplete understanding of rubiscos catalytic mechanism. Here, we report an atomic resolution (1.25 [A]) cryo-EM structure of spinach rubisco in complex with the transition-state analogue 2-carboxyarabinitol-1,5-bisphosphate. Supported by large-scale quantum/classical (QM/MM) calculations, our structural analysis reveals protonation equilibria within the active site and unexpected structural flexibility across large protein regions despite the exceptionally tight ligand binding. Our findings provide new insight into the complex interplay of protonation equilibria and conformational sampling, suggesting a novel basis for rubiscos rational redesign utilizing strategies that rely on a combination of dynamic and electrostatic control.

biochemistry↗

bioRxiv2025

Structure of active methyl-CoM reductase, Earth's main methane producer

Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the {beta}-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes.

biochemistry↗

bioRxiv2024

Precise Instruction and Consideration of the Vertical and Horizontal Force Component Increase Validity and Reliability of the 90:20 Isometric Posterior Chain Test

Hamstring injuries are associated with decreased hamstring strength. Matinlauri et al.s 90:20 Isometric Posterior Chain Test (90:20 IPCT) efficiently assesses hamstring strength, but has not been validated so far. Furthermore, their rather unprecise original instruction allows high variability in test execution. We added a new instruction and parameters and examined, whether this measure leads to increased reliability and validity. We assessed hamstring strength of 23 sport students via the 90:20 IPCT under the original instruction, to exert vertical force, and our new instruction, to exert vertical and horizontal force. Instead of only using bare vertical force as outcome parameter under the original (Fz_V) and our new instruction (Fz_VH), we also calculated the resultant force (Fres_VH) and the applied torque onto the force place (M_F_ortho_VH). To test for validity, we correlated the outcome parameters with peak torque of gold standard dynamometry. Furthermore, we measured muscle activities of the mm. rectus femoris, biceps femoris, semitendinosus, and gluteus maximus under our new instruction and compared them to those under the original parameter (Fz_V) via one sample t-tests. To evaluate reliability, tests were repeated on two separate days, for which we calculated intra class correlation coefficients (ICCs) and coefficients of variation (CVs). Our new instruction and parameters (Fz_VH, Fres_VH, M_F_ortho_VH) showed better validity (mean r = 0.77, r = 0.81, and r = 0.85) and equally good or better reliability (ICCs: 0.87, 0.89, and 0.94; CVs: 4.7 %, 4.1 %, and 4.7 %) than the original instruction and parameter (Fz_V) (mean r = 0.70; ICC: 0.91; CV: 5.6 %). There were no differences in muscle activities between the parameters and instructions of the 90:20 IPCT. We recommend our new instruction and the applied torque onto the force plate as it makes the 90:20 IPCT a more reliable and valid tool to assess hamstring strength.

physiology↗

bioRxiv2022

Redox-controlled structural reorganization and flavin strain within the ribonucleotide reductase R2b-NrdI complex monitored by serial femtosecond crystallography

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate- flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free-electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. We show that the flavin in the hydroquinone state is under steric strain in the R2b-NrdI protein complex, presumably tuning its redox potential to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2-interacton site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b- NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

biochemistry↗

bioRxiv2021

Structure Learning for Hierarchical Regulatory Networks

Network analysis offers a powerful technique to model the relationships between genes within biological regulatory networks. Inference of biological network structures is often performed on high-dimensional data, yet is hindered by the limited sample size of high throughput "omics" data typically available. To overcome this challenge, we exploit known organizing principles of biological networks that are sparse, modular, and likely share a large portion of their underlying architecture. We present SHINE - Structure Learning for Hierarchical Networks - a framework for defining data-driven structural constraints and incorporating a shared learning paradigm for efficiently learning multiple networks from high-dimensional data. We show through simulations SHINE improves performance when relatively few samples are available and multiple networks are desired, by reducing the complexity of the graphical search space and by taking advantage of shared structural information. We evaluated SHINE on TCGA Pan-Cancer data and found learned tumor-specific networks exhibit expected graph properties of real biological networks, recapture previously validated interactions, and recapitulate findings in literature. Application of SHINE to the analysis of subtype-specific breast cancer networks identified key genes and biological processes for tumor maintenance and survival as well as potential therapeutic targets for modulating known breast cancer disease genes.

bioinformatics↗

bioRxiv2021

Experimental evidence for delayed post-conflict management behaviour in wild dwarf mongooses

In many species, within-group conflict leads to immediate avoidance of potential aggressors or increases in affiliation, but no studies have investigated delayed post-conflict management behaviour. Here, we experimentally test that possibility using wild dwarf mongooses (Helogale parvula). First, we used natural and playback-simulated foraging displacements to demonstrate that bystanders take notice of the vocalisations produced during such within-group conflict events. We then used another playback experiment to assess delayed effects of within-group conflict on grooming interactions. Overall, fewer individuals groomed on evenings following an afternoon of simulated conflict, but those that did groomed more than on control evenings. Subordinate bystanders groomed with the simulated aggressor significantly less, and groomed more with one another, on conflict compared to control evenings. Our study provides experimental evidence that dwarf mongooses acoustically obtain information about within-group contests (including protagonist identity), retain that information and use it to inform conflict-management decisions with a temporal delay.

zoology↗

bioRxiv2020

Proteomic fingerprint identification of Neotropical hard tick species (Acari: Ixodidae) using a self-curated mass spectra reference library

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used as biomarkers for taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both principal component analysis (PCA) and linear discriminant analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens.Author Summary Hard ticks (Ixodidae) are external parasites that feed on the blood of almost every species of terrestrial vertebrate on earth, including humans. Due to a complete dependency on blood, both sexes and even immature stages, are capable of transmitting disease agents to their hosts, causing distress and sometimes death. Despite the public health significance of ixodid ticks, accurate species identification remains problematic. Vector species identification is core to developing effective vector control schemes. Herein, we provide the first report of MALDI identification of several species of field-collected Neotropical tick specimens preserved in ethanol for up to four years. Our methodology shows that identification does not depend on a commercial reference library of lab-reared samples, but with the help of machine learning it can rely on a self-curated reference library. In addition, our approach offers greater accuracy and lower cost per sample than conventional and modern identification approaches such as morphology and molecular barcoding.Competing Interest StatementThe authors have declared no competing interest.List of abbreviationsMALDImatrix-assisted laser desorption/ionizationPCAprincipal component analysisLDAlinear discriminant analysisDNAdeoxyribonucleic acidINDICASATInstitute for Scientific Research and High Technology ServicesSTRISmithsonian Tropical Research InstituteSNINational System of InvestigationUTPTechnological University of PanamaTOFtime-of-flightMiAmbienteMinistry of Environment.View Full Text

zoology↗

bioRxiv2017

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di--oxo bridged pair of Mn(III) ions, as an assembly intermediate.

biochemistry↗