Biology subjects

Martens, K.

Publications and source records attributed to Martens, K..

13 recordsLinked to original sources

bioRxiv2025

A novel expression system for imaging single-molecule fluorescence in Haloferax volcanii WR806 enables visualization of Cas1 involvement in UV-light associated DNA repair

Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and molecular biochemistry in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel expression system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins -- from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1 -- while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the systems utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal significant, dose-dependent changes in Cas1 mobility following UV-light induced damage, providing evidence for its involvement in DNA repair processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.

microbiology↗

bioRxiv2024

Lacticaseibacillus rhamnosus GG in a chewable colonizes the nose and facilitates local immune benefits in allergic rhinoconjunctivitis patients

Current treatments fall short in managing allergic rhinitis (AR), emphasizing the need for additional strategies. Beneficial bacteria application shows promise in AR, however most studies focus on oral probiotic administration without monitoring the applied strains in the upper respiratory tract (URT) and their local effects. In this randomized, double-blind, placebo-controlled trial, the probiotic Lacticaseibacillus rhamnosus GG was administered via chewable tablets in seasonal AR patients, randomized to probiotic (n=33) or placebo (n=31) groups. Per-protocol analysis of the URT microbiome, immune markers and AR symptoms was performed. L. rhamnosus GG trafficked from chewables to the oropharynx (77%, p=0.02) and nasopharynx (41%, p<0.0001). Control of self-reported AR symptoms via validated questionnaires under grass pollen exposure was observed after two weeks of probiotic administration and not upon placebo. A local decrease in salivary interleukin-4 (p < 0.05) and nasal IL-13 (p < 0.0001) was observed in the probiotic group. These data indicate that L. rhamnosus GG chewables can target the URT and exert local effects on key allergy cytokines after temporal probiotic engraftment. ImportanceAllergic rhinitis (AR) or hay fever is a highly prevalent condition, impacting nearly half the population in some countries. Supplementation of beneficial bacteria or probiotics has gained increasing attention in AR, and a key innovative way to do this is direct administration to the upper airways. Our study shows for the first time that the model probiotic strain Lacticaseibacillus rhamnosus GG can traffic to the nose in AR patients when administered via a slow-releasing chewable tablet. This trafficking is associated with local benefits in the airways, including on grass pollen-induced nasal symptoms and allergy-related cytokines.

microbiology↗

bioRxiv2023

Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

biophysics↗

bioRxiv2022

Single molecule long-read real-time amplicon-based sequencing of CYP2D6: a proof-of-concept with hybrid haplotypes

CYP2D6 is a widely expressed human xenobiotic metabolizing enzyme, best known for its role in the hepatic phase I cytochrome P450 enzyme system, where it metabolizes [~]20% of medications. It is also expressed in other organs including the brain, where its potential role in physiology and mental health traits and disorders is under further investigation. Owing to the presence of homologous pseudogenes in the CYP2D locus and transposable repeat elements in the intergenic regions, the gene encoding the CYP2D6 enzyme, CYP2D6, is one of the most hypervariable known human genes - with more than 165 core haplotypes. Haplotypes include structural variants, with a subtype of these known as hybrid haplotypes or fusion genes comprising part of CYP2D6 and part of its adjacent pseudogene, CYP2D7. The fusion genes are particularly challenging to identify. High fidelity (HiFi) single molecule real-time (SMRT) long-read sequencing can cover whole CYP2D6 haplotypes in a single continuous sequence, and is therefore ideal for structural variant detection. In addition, it is highly accurate and suitable for novel haplotype identification, which is necessary as new CYP2D6 haplotypes are continuously being discovered, and many more likely remain to be identified in relatively understudied populations such as Indigenous Peoples. The aim of the present work was to develop an efficient and accurate HiFi SMRT amplicon-based method capable of detecting the full range of CYP2D6 haplotypes including fusion genes. We report proof-of-concept for 24 amplicons including three positive controls, aligned to fusion gene haplotypes, with prior cross-validation data. Amplicons with CYP2D7-D6 fusion genes, including positive controls, aligned to the *13 subhaplotypes predicted (*13F, *13A2) with 100% accuracy, with the exception of one that aligned at 99.9%. Alignment of the *68 was 100% and above 99.9% to the CYP2D6*68 partial sequences EU5300606 and JF307779, respectively. The best alignments for the remaining CYP2D6-2D7 fusion genes were [≥]99.7% (to 3 significant figures). Lower percentage alignment for CYP2D6-2D7 fusion genes may reflect imperfect PCR optimization and/or the possibility that we may have haplotypes not yet in public databases. Further work on these is in progress. Moreover, we have adapted this method for non-hybrid haplotypes. This technique could therefore suffice for the characterization of the full range of CYP2D6 haplotypes. The method that we have developed could be extended to other complex loci and to other species in a multiplexed high throughput assay.

genomics↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2021

Commensal Pseudomonas fluorescens protect Arabidopsis from closely-related Pseudomonas pathogens in a colonization-dependent manner

Plants form commensal associations with soil microorganisms, creating a root microbiome that provides benefits to the host including protection against pathogens. While bacteria can inhibit pathogens through production of antimicrobial compounds in vitro, it is largely unknown how microbiota contribute to pathogen protection in planta. We developed a gnotobiotic model system consisting of Arabidopsis thaliana, and an opportunistic pathogen Pseudomonas sp. N2C3, to identify mechanisms that determine the outcome of plant-pathogen-microbiome interactions in the rhizosphere. We screened 25 phylogenetically diverse Pseudomonas strains for their ability to protect against N2C3 and found that commensal strains closely related to N2C3 were more likely to protect against pathogenesis. We used a comparative genomics approach to identify unique genes in the protective strains that revealed no genes that correlate with protection, suggesting that variable regulation of components of the core Pseudomonas genome may contribute to pathogen protection. We found that commensal colonization level was highly predictive of protection and so tested deletions in genes previously shown to be required for Arabidopsis rhizosphere colonization. We identified a response regulator colR that is required for Pseudomonas protection from N2C3 and fitness in competition with N2C3 indicating that competitive exclusion may contribute to pathogen protection. We found that Pseudomonas WCS365 also protects against the agricultural pathogen Pseudomonas fuscovaginae SE-1, the causal agent of bacterial sheath brown rot of rice. This work establishes a gnotobiotic model to uncover mechanisms by which members of the microbiome can protect hosts from pathogens and informs our understanding of the use of beneficial strains for microbiome engineering in dysbiotic soil systems.

microbiology↗

bioRxiv2021

Characterization of a novel variant in the HR1 domain of MFN2 in a patient with ataxia, optic atrophy and sensorineural hearing loss

Pathogenic variants in MFN2 cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some MFN2 variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between MFN2 and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic MFN2 variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions. Here, we describe a novel homozygous MFN2 variant, D414V, in a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy. Characterization of patient fibroblasts reveals phenotypes consistent with impaired MFN2 functions and expands the phenotypic presentation of MFN2 variants to include cerebellar ataxia.

genetics↗

bioRxiv2020

First annotated draft genomes of non-marine ostracods (Ostracoda, Crustacea) with different reproductive modes

ABSTRACTSOstracods are one of the oldest crustacean groups with an excellent fossil record and high importance for phylogenetic analyses but genome resources for this class are still lacking. We have successfully assembled and annotated the first reference genomes for three species of non-marine ostracods; two with obligate sexual reproduction (Cyprideis torosa and Notodromas monacha) and the putative ancient asexual Darwinula stevensoni. This kind of genomic research has so far been impeded by the small size of most ostracods and the absence of genetic resources such as linkage maps or BAC libraries that were available for other crustaceans. For genome assembly, we used an Illumina-based sequencing technology, resulting in assemblies of similar sizes for the three species (335-382Mb) and with scaffold numbers and their N50 (19-56 kb) in the same orders of magnitude. Gene annotations were guided by transcriptome data from each species. The three assemblies are relatively complete with BUSCO scores of 92-96%, and thus exceed the quality of several other published crustacean genomes obtained with similar techniques. The number of predicted genes (13,771-17,776) is in the same range as Branchiopoda genomes but lower than in most malacostracan genomes. These three reference genomes from non-marine ostracods provide the urgently needed basis to further develop ostracods as models for evolutionary and ecological research.

evolutionary biology↗

bioRxiv2020

The SARS-CoV-2 and other human coronavirus spike proteins are fine-tuned towards temperature and proteases of the human airways

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV-1 and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. First, the SARS-CoV-2 spike (SARS-2-S) reached higher levels in pseudoparticles when produced at 33{degrees}C instead of 37{degrees}C. Even stronger preference for the upper airway temperature of 33{degrees}C was evident for the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV-1 and MERS-CoV favored 37{degrees}C, in accordance with their preference for the lower airways. Next, SARS-2-S proved efficiently activated by TMPRSS13, besides the previously identified host cell protease TMPRSS2, which may broaden the cell tropism of SARS-CoV-2. TMPRSS13 was found to be an effective spike activator for the virulent coronaviruses but not the common cold HCoV-229E virus. Activation by these proteases requires pre-cleavage of the SARS-2-S S1/S2 cleavage loop, and both its furin motif and extended loop length proved critical to achieve virus entry into airway epithelial cells. Finally, we show that the D614G mutation in SARS-2-S increases S protein stability and expression at 37{degrees}C, and promotes virus entry via cathepsin B/L activation. These spike properties might promote virus spread, potentially explaining why the G614 variant is currently predominating worldwide. Collectively, our findings indicate how the coronavirus spike protein is fine-tuned towards the temperature and protease conditions of the airways, to enhance virus transmission and pathology. SIGNIFICANCE STATEMENTThe rapid spread of SARS-CoV-2, the cause of COVID-19, is related to abundant replication in the upper airways, which is not observed for other highly pathogenic human coronaviruses. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards different parts of the respiratory tract. Coronavirus spikes exhibit distinct temperature preference to precisely match the upper (~33{degrees}C) or lower (37{degrees}C) airways. We identified airway proteases that activate the spike for virus entry into cells, including one protease that may mediate coronavirus virulence. Also, a link was seen between spike stability and entry via endosomal proteases. This mechanism of spike fine-tuning could explain why the SARS-CoV-2 spike-D614G mutant is more transmissible and therefore globally predominant.

microbiology↗

bioRxiv2019

Cross-validation of technologies for genotyping CYP2D6 and CYP2C19

BackgroundCYP2D6 and CYP2C19 are cytochrome P450 enzymes involved in the metabolism of many medications from multiple therapeutic classes. Associations between patterns of variants (known as haplotypes) in the genes encoding them (CYP2D6 and CYP2C19) and enzyme activities are well described. The genes in fact comprise 21% of biomarkers in drug labels. Despite this, genotyping is not common, partly attributable to its challenging nature (CYP2D6 having >100 haplotypes, including those with sequence from an adjacent pseudogene, and gene duplications). We cross-validated different methodologies for identifying haplotypes in these genes against each other. MethodsNinety-two samples with a variety of CYP2D6 and CYP2C19 genotypes according to prior AmpliChip CYP450 and TaqMan CYP2C19*17 data were selected from the Genome-based therapeutic drugs for depression (GENDEP) study. Genotyping was performed with TaqMan copy number variant (CNV) and single nucleotide variant (SNV) analysis, the next generation sequencing-based Ion S5 AmpliSeq Pharmacogenomics Panel, PharmacoScan, long-range polymerase chain reaction (L-PCR) followed by amplicon analysis, and Agena for CYP2C19. Variant pattern to haplotype translation was automated. ResultsThe inter-platform concordance for CYP2C19 was high (up to 100% for available data). For CYP2D6, the IonS5-PharmacoScan concordance was 94% for a range of variants tested apart from those with at least one extra copy of a CYP2D6 gene (occurring at a frequency of 3.8%, 33/853), or those with substantial sequence derived from pseudogene, known as hybrids (3%, 26/853). ConclusionsInter-platform concordance for CYP2C19 was high, and, moreover, the Ion S5 and PharmacoScan data were 100% concordant with that from a TaqMan CYP2C19*2 assay. We have also demonstrated feasibility of using an NGS platform for genotyping CYP2D6 and CYP2C19, with automated data interpretation methodology. This points the way to a method of making CYP2D6 and CYP2C19 genotyping more readily accessible.

genetics↗

bioRxiv2019

Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism

CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA ([~]30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.\n\nOne sentence summaryThe results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.

microbiology↗

bioRxiv2018

Confinement-induced transition between wave-like collective cell migration modes

The structural and functional organization of biological tissues relies on the intricate interplay between chemical and mechanical signaling. Whereas the role of constant and transient mechanical perturbations is generally accepted, several studies recently highlighted the existence of long-range mechanical excitations (i.e., waves) at the supracellular level. Here, we confine epithelial cell mono-layers to quasi-one dimensional geometries, to force the establishment of tissue-level waves of well-defined wavelength and period. Numerical simulations based on a self-propelled Voronoi model reproduce the observed waves and exhibit a phase transition between a global and a multi-nodal wave, controlled by the confinement size. We confirm experimentally the existence of such a phase transition, and show that wavelength and period are independent of the confinement length. Together, these results demonstrate the intrinsic origin of tissue oscillations, which could provide cells with a mechanism to accurately measure distances at the supracellular level.

biophysics↗

bioRxiv2017

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): an algorithm for MHz localization rates using standard CPUs

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

biophysics↗