Biology subjects

Fuchs, J.

Publications and source records attributed to Fuchs, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org. Each genome was constructed using a novel assembly tool called Viridian (https://github.com/iqbal-lab-org/viridian), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

bioinformatics↗

bioRxiv2023

Chromosome-level genome assembly of the European Green woodpecker Picus viridis

The European Green Woodpecker, Picus viridis, is a widely distributed species found in the Western Palearctic region. Here we assembled a highly contiguous genome assembly for this species using a combination of short and long reads sequencing and scaffolded with chromatin conformation capture (Hi-C). The final genome assembly was 1.28 Gb and features a scaffold N50 of 37Mb and a scaffold L50 of 39.165 Mb. The assembly incorporates 89.4% of the genes identified in birds in OrthoDB. Gene and repetitive content annotation on the assembly detected 15,805 genes and a [~]30.1% occurrence of repetitive elements, respectively. Analysis of synteny demonstrates the fragmented nature of the Picus viridis genome when compared to the chicken (Gallus gallus). The assembly and annotations produced in this study will certainly help for further research into the genomics of P. viridis and the comparative evolution of woodpeckers.

genomics↗

bioRxiv2023

Steady-state neuron-predominant LINE-1 encoded ORF1p protein and LINE-1 RNA increase with aging in the mouse and human brain

Recent studies have established a reciprocal causal link between aging and the activation of transposable elements, characterized in particular by a de-repression of LINE-1 retrotransposons. These LINE-1 elements represent 21% of the human genome, but only a minority of these sequences retain the coding potential essential for their mobility. LINE-1 encoded proteins can induce cell toxicity implicated in aging and neurodegenerative diseases. However, our knowledge of the expression and localization of LINE-1-encoded proteins in the central nervous system is limited. Using a novel approach combining atlas-based brain mapping with deep-learning algorithms on large-scale pyramidal brain images, we unveil a heterogeneous, neuron-predominant and widespread ORF1p expression throughout the murine brain at steady-state. In aged mice, ORF1p expression increases significantly which is corroborated in human post-mortem dopaminergic neurons by an increase in young LINE-1 elements including those with open reading frames. Mass spectrometry analysis of endogenous mouse ORF1p revealed novel, neuron-specific protein interactors. These findings contribute to a comprehensive description of the dynamics of LINE-1 and ORF1p expression in the brain at steady-state and in aging and provide insights on ORF1p protein interactions in the brain.

neuroscience↗

bioRxiv2023

Combining CRISPR/Cas mediated terminal resolution with a novel genetic workflow to achieve high diversity adenoviral libraries

While recombinant Adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR/Cas9 mediated in vivo terminal resolution (CTR) aiding high efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of BAC-based rAd-libraries employing the efficiency of CTR. We utilized frequent, pre-existing genomic sequences to allow insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In a second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half-site directed fragment replacement (HFR) allows introduction of >106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of [~]2.5x104 modified rAd per cm2 of transfected cell culture. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/566979v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db61a7org.highwire.dtl.DTLVardef@1e85764org.highwire.dtl.DTLVardef@c17178org.highwire.dtl.DTLVardef@a299_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets

BackgroundSugar beet (Beta vulgaris subsp. vulgaris) and its crop wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNA sequences represent the fastest evolving parts of the genome, they likely impact genomic variability and contribute to the separation of beet gene pools. Hence, we investigated if innovations and losses in the repeatome can be linked to chromosomal differentiation and speciation. ResultsWe traced genome- and chromosome-wide evolution across sugar beet and twelve wild beets comprising all sections of the beet genera Beta and Patellifolia. For this, we combined data from short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive data framework for our beet panel that spans the complete scale from DNA sequence to chromosome up to the genome. Genome sizes and repeat profiles reflect the separation of the beet species into three gene pools. These gene pools harbor repeats with contrasting evolutionary patterns: We identified section- and species-specific repeat emergences and losses, e.g. of the retrotransposons causal for genome expansions in the section Corollinae/Nanae. Since most genomic variability was found in the satellite DNAs, we focused on tracing the 19 beetSat families across the three beet sections/genera. These taxa harbor evidence for contrasting strategies in repeat evolution, leading to contrasting satellite DNA profiles and fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia species to the formation of patchwork chromosomes in Corollinae/Nanae species. ConclusionsWe show that repetitive DNA sequences are causal for genome size expansion and contraction across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably among beet taxa, leading to the evolution of distinct chromosomal setups. These differences likely contribute to the barriers in beet breeding between the three gene pools. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genome variability, and chromosomal differentiation/evolution and provide a theoretical basis for understanding barriers in crop breeding.

genomics↗

bioRxiv2023

Nuclear translocation of LINE-1 ORF1p alters nuclear envelope integrity and disrupts nucleocytoplasmic transport in neurons

LINE-1 retrotransposons are emerging as possible culprits in neurodegenerative diseases. However, the molecular mechanisms underlying the pathogenic role of LINE-1 and their encoded proteins ORF1p and ORF2p are still not completely understood. While the endonuclease and reverse transcriptase activities of ORF2p have been associated with DNA damage and inflammation, no pathogenic role has yet been assigned to ORF1p. Using a neuronal model of oxidative stress displaying increased LINE-1 expression, we report here that ORF1p stress-dependently translocated into the nucleus, localized to the nuclear envelope and directly interacted with nuclear import proteins, nuclear pore complex components and the inner nuclear lamina. Stress-dependent targeting of nuclear envelope components by ORF1p altered nuclear envelope integrity, disrupted nucleocytoplasmic transport and induced heterochromatin destructuration, features associated with neurodegenerative diseases and aging. Neurons of post-mortem Parkinson disease (PD) patients and non-PD affected controls expressed ORF1p and nuclear ORF1p levels correlated with altered nuclear shape in PD. Overexpression of ORF1p in neurons in the absence of stress recapitulated nuclear envelope dysfunctions and increased nuclear ORF1p levels correlated with a loss of nuclear circularity. Stress-induced nuclear alterations were restored by blocking ORF1p nuclear import or by the small molecule remodelin. This study thus reveals a retrotransposition- and ORF2p- independent pathogenic action of ORF1p at the nuclear envelope and points to ORF1p as a novel target for neuroprotection.

neuroscience↗

bioRxiv2022

What doesn't kill you makes you live longer - Longevity of a social host linked to parasite proteins

Parasites with complex lifecycles often manipulate the phenotype of their intermediate hosts to increase the probability of transmission to their definitive hosts. Infection with Anomotaenia brevis, a cestode that uses Temnothorax nylanderi ants as intermediate hosts, leads to a multiple-fold extension of host lifespan and to changes in behaviour, morphology, and colouration. The mechanisms behind these changes are unknown, as is whether the increased longevity is achieved through parasite manipulation. Here we demonstrate that the parasite releases proteins into its host with functions that might explain the observed changes. These parasitic proteins make up a substantial portion of the proteome of the hosts haemolymph, and thioredoxin peroxidase and superoxide dismutase, two antioxidants, exhibited the highest abundances among them. The largest part of the secreted proteins could not be annotated, indicating they are either novel or severely altered during recent coevolution to function in host manipulation. We also detected shifts in the hosts proteome with infection, in particular an overabundance of vitellogenin-like-A in infected ants, a protein that regulates division of labour in Temnothorax ants, which could explain the observed behavioural changes. Our results thus point at two different strategies likely employed by this parasite to manipulate its host - by secretion of proteins with immediate influence on the hosts phenotype and by altering the hosts translational activity. Our findings reveal the intricate molecular interplay required to influence the phenotype of a host and shed light on potential signalling pathways and genes involved in parasite-host communication.

evolutionary biology↗

bioRxiv2022

Plasticity in centromere organization: Holocentromeres can consist of merely a few megabase-sized satellite arrays

The centromere is the chromosome region where the microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere location, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly-spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms distinctly clustered centromeres in chromocenters at interphase. Additionally, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Using polymer simulations, we modeled the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about the diversity of centromere organization, showing that holocentricity is not restricted to species with numerous and small centromere units.

plant biology↗

bioRxiv2022

Lung macrophages utilize cathepsin K-dependent phagosomal machinery to degrade intracellular collagen

Resident tissue macrophages (RTMs) are organ-specialized phagocytes responsible for the maintenance and protection of tissue homeostasis. It is well established that tissue diversity is reflected by the heterogeneity of RTMs origin and phenotype. However, much less is known about tissue-specific phagocytic and proteolytic macrophage functions. Here, using quantitative proteomics approach, we identify cathepsins as key determinants of phagosome maturation in primary peritoneal, lung and brain resident macrophages. The data further uncover cathepsin K (CtsK) as a molecular marker for lung phagosomes required for intracellular protein and collagen degradation. Pharmacological blockade of CtsK activity diminished phagosomal proteolysis and collagenolysis in lung resident macrophages. Furthermore, pro-fibrotic TGF-{beta} negatively regulated CtsK-mediated phagosomal collagen degradation independently from classical endocytic proteolytic pathways. In humans, phagosomal CtsK activity was reduced in COPD lung macrophages and non-COPD lung macrophages exposed to cigarette smoke extract. Taken together, this study provides a comprehensive map of how peritoneal, lung and brain tissue environment shapes phagosomal composition, revealing CtsK as a key molecular determinant of lung phagosomes contributing to phagocytic collagen clearance in lungs.

cell biology↗

bioRxiv2022

Impaired immune response drives age-dependent severity of COVID-19

SARS-CoV-2 is a highly contagious respiratory virus and the causative agent for COVID-19. The severity of disease varies from mildly symptomatic to lethal and shows an extraordinary correlation with increasing age, which represents the major risk factor for severe COVID-191. However, the precise pathomechanisms leading to aggravated disease in the elderly are currently unknown. Delayed and insufficient antiviral immune responses early after infection as well as dysregulated and overshooting immunopathological processes late during disease were suggested as possible mechanisms. Here we show that the age-dependent increase of COVID-19 severity is caused by the disruption of a timely and well-coordinated innate and adaptive immune response due to impaired interferon (IFN) responses. To overcome the limitations of mechanistic studies in humans, we generated a mouse model for severe COVID-19 and compared the kinetics of the immune responses in adult and aged mice at different time points after infection. Aggravated disease in aged mice was characterized by a diminished IFN-{gamma} response and excessive virus replication. Accordingly, adult IFN-{gamma} receptor-deficient mice phenocopied the age-related disease severity and supplementation of IFN-{gamma} reversed the increased disease susceptibility of aged mice. Mimicking impaired type I IFN immunity in adult and aged mice, a second major risk factor for severe COVID-192-4, we found that therapeutic treatment with IFN-{lambda} in adult and a combinatorial treatment with IFN-{gamma} and IFN-{lambda} in aged Ifnar1-/-mice was highly efficient in protecting against severe disease. Our findings provide an explanation for the age-dependent disease severity of COVID-19 and clarify the nonredundant antiviral functions of type I, II and III IFNs during SARS-CoV-2 infection in an age-dependent manner. Based on our data, we suggest that highly vulnerable individuals combining both risk factors, advanced age and an impaired type I IFN immunity, may greatly benefit from immunotherapy combining IFN-{gamma} and IFN-{lambda}.

immunology↗

bioRxiv2022

High temperature increases centromere-mediated genome elimination frequency in Arabidopsis deficient in cenH3 or its assembly factor KNL2

Double haploid production is the most effective way of creating true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when outcrossed to wild-type. Here we report that a mutant of the cenH3 assembly factor KNL2 can be used as a haploid inducer. We elucidated that short temperature stress of the knl2 mutant increased the efficiency of haploid induction from 1 to 10%. Moreover, we have demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid inducing lines, suggesting that haploid inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the GMO approach at any stage. In addition, we have shown that the cenh3-4 mutant, which does not induce haploids under standard growth conditions, functions as a haploid inducer after exposure to short temperature stress.

plant biology↗

bioRxiv2022

Automated Analysis of Neuronal Morphology through an Unsupervised Classification Model of Neurites

Brain function emerges from a highly complex network of specialized cells that are interlinked by billions of synapses. The synaptic connectivity between neurons is established between the elongated processes of their axons and dendrites or, together, neurites. To establish these billions of often far-reaching connections, cellular neurites have to grow in highly specialized, cell-type dependent patterns covering often mm distances and connecting with thousands of other neurons. The outgrowth and branching of neurites are tightly controlled during development and are a commonly used functional readout of imaging in the neurosciences. Manual analysis of neuronal morphology from microscopy images, however, is very time intensive and error prone. Especially fully automated segmentation and classification of all neurites remain unavailable in open-source software. Here we present a standalone, GUI-based software for batch-quantification of neuronal morphology in fluorescence micrographs with minimal requirements for user interaction. Neurons are segmented using a Hessian-based algorithm to detect thin neurite structures combined with intensity- and shape-based detection of the cell body. To measure the number of branches in a neuron accurately, rather than just determining branch points, neurites are classified into axon, dendrites and their branches of increasing order by their length using a geodesic distance transform of the cell skeleton. The software was benchmarked against a large, published dataset and reproduced the phenotype observed after manual annotation before. Our tool promises greatly accelerated and improved morphometric studies of neuronal morphology by allowing for consistent and automated analysis of large datasets.

bioinformatics↗

bioRxiv2021

GiPS: Genomics-informed parent selection uncovers the breeding value of wheat genetic resources

The great efforts spent in the maintenance of past diversity in genebanks are rationalized by the potential role of plant genetic resources in future crop improvement - a concept whose practical implementation has fallen short of expectations. Here, we implement genomics-informed parent selection to expedite pre-breeding without discriminating against non-adapted germplasm. We collect dense genetic profiles for a large winter wheat collection and evaluate grain yield and resistance to yellow rust in representative coresets. Genomic prediction within and across genebanks identified the best parents for PGR x elite derived crosses that outyielded current elite cultivars in multiple field trials.

genomics↗

bioRxiv2021

Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization

Several effective SARS-CoV-2 vaccines are currently in use, but in the light of waning immunity and the emergence of novel variants, effective boost modalities are needed in order to maintain or even increase immunity. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic DNA or mRNA priming result in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with an mRNA vaccine, the mucosal boost with adenoviral vectors induced high levels of IgA and tissue-resident memory T cells in the respiratory tract. Mucosal neutralization of virus variants of concern was also enhanced by the intranasal boosts. Importantly, priming with mRNA provoked a more comprehensive T cell response consisting of circulating and tissue-resident memory T cells after the boost, while a DNA priming induced mostly mucosal T cells. Concomitantly, the intranasal boost strategies provided protection against symptomatic disease. Therefore, a mucosal booster immunization after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.

immunology↗

bioRxiv2021

Precursor types predict the stability of neuronal branches

Branches are critical for neuron function, generating the morphological complexity required for functional networks. They emerge from different, well-described, cytoskeletal precursor structures that elongate to branches. While branches are thought to be maintained by shared cytoskeletal regulators, our data from mouse hippocampal neurons indicate that the precursor structures trigger alternative branch maintenance mechanisms with differing stabilities. While branches originating from lamellipodia or growth cone splitting events collapse soon after formation, branches emerging from filopodia persist. Furthermore, compared to other developing neurites, axons stabilise all branches and preferentially initiate branches from filopodia. These differences explain the decreased stability of branches we observe in neurons lacking the plasma membrane protein phospholipid phosphatase related protein 3 (PLPPR3/PRG2). Rather than altering branch stability directly, PLPPR3 boosts a filopodia branch program on axons, thereby indirectly initiating more long-lived branches. We propose that studies on branching should distinguish overall stabilising effects from effects on precursor types, ideally using multifactorial statistical models as exemplified in this study.

cell biology↗

bioRxiv2020

The landscape of SARS-CoV-2 RNA modifications

In 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the first documented cases of severe lung disease COVID-19. Since then, SARS-CoV-2 has been spreading around the globe resulting in a severe pandemic with over 500.000 fatalities and large economical and social disruptions in human societies. Gaining knowledge on how SARS-Cov-2 interacts with its host cells and causes COVID-19 is crucial for the intervention of novel therapeutic strategies. SARS-CoV-2, like other coronaviruses, is a positive-strand RNA virus. The viral RNA is modified by RNA-modifying enzymes provided by the host cell. Direct RNA sequencing (DRS) using nanopores enables unbiased sensing of canonical and modified RNA bases of the viral transcripts. In this work, we used DRS to precisely annotate the open reading frames and the landscape of SARS-CoV-2 RNA modifications. We provide the first DRS data of SARS-CoV-2 in infected human lung epithelial cells. From sequencing three isolates, we derive a robust identification of SARS-CoV-2 modification sites within a physiologically relevant host cell type. A comparison of our data with the DRS data from a previous SARS-CoV-2 isolate, both raised in monkey renal cells, reveals consistent RNA modifications across the viral genome. Conservation of the RNA modification pattern during progression of the current pandemic suggests that this pattern is likely essential for the life cycle of SARS-CoV-2 and represents a possible target for drug interventions.

genetics↗

bioRxiv2020

The structure of enteric human adenovirus 41 - a leading cause of diarrhoea in children

Human adenovirus (HAdV) types F40 and F41 are a prominent cause of diarrhea and diarrhea-associated mortality in young children worldwide. These enteric HAdVs differ strikingly in tissue tropism and pathogenicity from respiratory and ocular adenoviruses, but the structural basis for this divergence has been unknown. Here we present the first structure of an enteric HAdV - HAdV-F41 - determined by cryo-EM to a resolution of 3.8[A]. The structure reveals extensive alterations to the virion exterior as compared to non-enteric HAdVs, including a unique arrangement of capsid protein IX. The structure also provides new insights into conserved aspects of HAdV architecture such as a proposed location of protein V, which links the viral DNA to the capsid, and assembly-induced conformational changes in the penton base protein. Our findings provide the structural basis for adaptation to a fundamentally different tissue tropism of enteric HAdVs.

microbiology↗

bioRxiv2020

Perturbed DNA methylation by sustained overexpression of Gadd45b induces chromatin disorganization, DNA strand breaks and dopaminergic neuron death in mice

Heterochromatin disorganization is a key hallmark of aging and DNA methylation state is currently the main molecular predictor of chronological age. The most frequent neurodegenerative diseases like Parkinson disease and Alzheimers disease are age-related but how the aging process and chromatin alterations are linked to neurodegeneration is unknown. Here, we investigated the consequences of viral overexpression of Gadd45b, a multifactorial protein involved in active DNA demethylation, in the midbrain of wild-type mice. Gadd45b overexpression induces global and stable changes in DNA methylation, particularly on gene bodies of genes related to neuronal functions. DNA methylation changes were accompanied by perturbed H3K9me3-marked heterochromatin and increased DNA damage. Prolonged Gadd45b expression resulted in dopaminergic neuron degeneration accompanied by altered expression of candidate genes related to heterochromatin maintenance, DNA methylation or Parkinson disease. Gadd45b overexpression rendered midbrain dopaminergic neurons more vulnerable to acute oxidative stress. Heterochromatin disorganization and DNA demethylation resulted in derepression of mostly young LINE-1 transposable elements, a potential source of DNA damage, prior to Gadd45b-induced neurodegeneration. Our data implicate that alterations in DNA methylation and heterochromatin organization, LINE-1 derepression and DNA damage can represent important contributors in the pathogenic mechanisms of dopaminergic neuron degeneration with potential implications for Parkinson disease.

neuroscience↗