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Stark, M.

Publications and source records attributed to Stark, M..

11 recordsLinked to original sources

Stable, intronic RNAs explain preservation of introns in Cyanidioschyzon merolae

Despite recent work identifying functional roles for introns, we lack a broad understanding of why some introns are preserved while others are removed. Here, we use the thermophilic red alga, Cyanidioschyzon merolae, as a model to investigate why only 39 of the approximately 2000 ancestral introns were preserved in this lineage. We observe that 23 of the 39 introns encode stable RNAs, 11 of which represent a novel class of non-coding RNA (ncRNA), which we call stable intronically-encoded RNAs (sieRNAs). These novel ncRNAs are expressed constitutively under normal growth conditions and are conserved in other extremophilic algae. One sieRNA, Q270, is predicted to stabilize a chloroplast polycistronic transcript encoding 17 ribosomal protein genes through direct antisense base pairing. Strikingly, all sieRNAs are polyadenylated under heat stress with long tails ranging from 50-200 nucleotides long, linking them to a potential heat stress response that may be critical for heat adaptation. Furthermore, DMS-MaP chemical probing revealed that some sieRNAs contain three-way junctions, a common RNA regulatory element, while others undergo accessibility shifts between in vivo and in vitro conditions, indicative of cellular interactions. Our findings suggest that introns in C. merolae are preserved to encode ncRNAs and suggest that introns may serve as hosts for regulatory RNAs across eukaryotes.

molecular biology↗

Disrupted Forward Connectivity in Parieto-temporal Network Impairs Memory Performance in Alzheimer's Disease

Alzheimers disease (AD) is characterised by the accumulation of beta-amyloid (A{beta}) and tau proteins, leading to neurodegeneration and cognitive decline. While A{beta} and tau are known to disrupt synaptic function, the mechanisms linking these molecular pathologies to network-level dysfunction and memory impairment remain poorly understood. Here we investigated the effects of A{beta} and tau pathology (CSF A{beta}42/40 ratio and tau phosphorylated at position 181, p-tau-181, respectively) on effective connectivity (EC) related to memory encoding, which may constitute a link between synaptic pathology and cognitive outcomes. Functional magnetic resonance imaging (fMRI) during visual memory encoding was acquired from participants of the multicentric DZNE Longitudinal Cognitive Impairment and Dementia Study (DELCODE), including 203 cognitively normal older participants (CN) as well as individuals with subjective cognitive decline (SCD; N = 204), mild cognitive impairment (MCI; N = 65), and early dementia due to AD (DAT; N = 21). EC was assessed by applying Dynamic causal modelling (DCM) to the fMRI data, using brain regions previously implicated in memory-encoding: the parahippocampal place area (PPA), the hippocampus (HC) and the precuneus (PCU). Disruptions in forward connectivity from the PPA to the HC and PCU were associated with both memory impairment and indices of AD pathology. Specifically, reduced excitatory EC from the PPA to the HC was associated with higher p-tau-181 levels and correlated with poorer memory performance. Diminished inhibitory EC from the PPA to the PCU was driven by both tau and amyloid pathology and was likewise linked to memory decline. Our findings suggest that disrupted forward connectivity within the temporo-parietal memory network constitutes a candidate mechanism mediating the relationship between molecular pathology and cognitive dysfunction.

neuroscience↗

Structural basis of DNA recombination catalysis and regulation by phiC31 integrase

Large serine integrases catalyse the integration and excision of bacteriophage (phage) DNA genomes into and from the genomes of their bacterial hosts by site-specific recombination1. These recombination reactions are notable for their unidirectionality: integrase recognises and efficiently recombines short (40-50-bp) sequences in the phage (attP) and bacterial (attB) genomes, but is inactive on the product sites attL and attR flanking the inserted prophage, ensuring stable integration. The reverse reaction (excision) only occurs in the presence of a second phage-encoded protein, the Recombination Directionality Factor (RDF). Their strict recombination directionality has made serine integrases versatile tools in emerging genome-editing technologies2-5; however, the structural basis of directionality remains. Here we report structures of {phi}C31 integrase, the most-studied and most widely used member of the serine integrase family, in complexes with its DNA recombination sites, with and without its RDF. These structures correspond to four key mechanism steps: integration of the phage DNA genome (attP x attB); excision of the integrated prophage DNA, mediated by integrase and RDF (attL x attR); inhibition of integrase-catalysed excision in the absence of RDF (attL dimer complex); and inhibition of integrase-catalysed integration in the presence of RDF (attB dimer complex). Our data provide a mechanistic understanding of how the serine integrase recombination system establishes and regulates phage lysogeny, and lay the foundation for future development of integrase-based genome-editing technologies.

biochemistry↗

SMAS: Structural MRI-Based AD Score using Bayesian VAE

This study introduces the Structural MRI-based Alzheimers Disease Score (SMAS), a novel index intended to quantify Alzheimers Disease (AD)-related morphometric patterns using a deep learning Bayesian-supervised Variational Autoencoder (Bayesian-SVAE). SMAS index was constructed using baseline structural MRI data from the DELCODE study and evaluated longitudinally in two independent cohorts: DEL-CODE (n=415) and ADNI (n=190). Our findings indicate that SMAS has strong associations with cognitive performance (DELCODE: r=-0.83; ADNI: r=-0.62), age (DEL-CODE: r=0.50; ADNI: r=0.28), hippocampal volume (DEL-CODE: r=-0.44; ADNI: r=-0.66), and total grey matter volume (DELCODE: r=-0.42; ADNI: r=-0.47), suggesting its potential as a biomarker for AD-related brain atrophy. Moreover, our longitudinal studies suggest that SMAS may be useful for early identification and tracking of AD. The model demonstrated significant predictive accuracy in distinguishing cognitively healthy individuals from those with AD (DELCODE: AUC=0.971 at baseline, 0.833 at 36 months; ADNI: AUC=0.817 at baseline, improving to 0.903 at 24 months). Notably, over a 36-month period, SMAS index outperformed existing measures such as SPARE-AD and hippocampal volume. Relevance map analysis revealed significant morphological changes in key AD-related brain regions--including the hippocampus, posterior cingulate cortex, precuneus, and lateral parietal cortex--highlighting that SMAS is a sensitive and interpretable biomarker of brain atrophy, suitable for early AD detection and longitudinal monitoring of disease progression.

neuroscience↗

Dysfunction of the episodic memory network in the Alzheimer's disease cascade

Alzheimers disease (AD) is a major cause of dementia and cognitive decline. Here we assessed how episodic memory circuit dysfunction, a hallmark of AD, is related to the longitudinal cascade of AD biomarkers, neurodegeneration and cognition using data from the DZNE Longitudinal Cognitive Impairment and Dementia study. This data set is unique by including over 1000 longitudinal functional magnetic resonance imaging (fMRI) measurements during episodic memory encoding. We leveraged a disease progression model (DPM) to obtain AD progression scores. Voxel-wise analyses revealed widespread loss of deactivation (hyperactivation) and activation (hypoactivation) with increasing disease stage. Hyperactivation trajectories were nonlinear and visually preceded trajectories of cognition. Overall, hyperactivation was independently associated with co-occurrence of amyloid- and tau-positivity and neurodegeneration, suggesting synaptic dysfunction and neurodegeneration as two independent drives of cognitive decline. Our results therefore provide evidence for a critical time window in which pharmacological treatments targeting the synapse may improve cognition.

neuroscience↗

Molecular mechanisms of long-term light adaptation of an extremophilic alga Cyanidioschyzon merolae

Oxygenic phototrophs have evolved a remarkable plethora of strategies to react to changes in light intensity and spectral range, which allows them to thrive in a wide range of environmental conditions. Varying light quality and quantity influences the balance between solar energy capture and utilisation in photosynthesis, affecting concomitantly the downstream processes of central carbon and nitrogen metabolism as well as cellular growth and division. Here, we performed a comprehensive analysis of the mechanisms of long-term photoacclimation of an extremophilic red alga Cyanidioschyzon merolae that grows in sulphuric hot springs at high temperatures and low pH. By using spectroscopic, confocal fluorescence microscopy, photosynthetic performance measurements and global transcriptome analyses, we identified several molecular mechanisms underlying the long-term adaptation of this acido-thermophilic red alga to varying light intensity and spectral quality. These include: (1) remodelling of the functional antenna size of both photosystems; (2) rearrangement of the PSB/PSII/PSI microdomains within thylakoids; (3) modulation of the photosynthetic performance parameters, especially at the level of non-photochemical quenching, and (4) transcriptional regulation of photosynthesis and its regulatory components as well as downstream metabolic pathways related to ROS detoxification, cell/organelle division, and central carbon and nitrogen metabolism. Such an intricate network of interplay between light-driven reactions and downstream metabolic pathways provides the necessary basis for maintaining the highest photosynthetic performance under light-limiting conditions.

plant biology↗

Interaction of habitat isolation and seasonality: impact of fragmentation in dynamic landscapes on local and regional population variability in meta-food chains

While habitat loss is a known key driver of biodiversity decline, the effect of habitat fragmentation per se is far less clear. Environmental perturbations, e.g. due to seasonality, potentially interact with effects of habitat fragmentation, thus making the latter even more difficult to assess. In order to analyze how the dynamic stability of local and metapopulations is affected by habitat fragmentation, we simulate a meta-food chain with three species on complex networks of habitat patches and evaluate the average variability of the local populations and the metapopulations as well as the level of synchronization between the patches. To account for environmental perturbations, we contrast simulations of static landscapes with simulations of dynamic landscapes, where 30 percent of the patches periodically become unavailable as habitats. We find that it depends on the parameterization of the food chain whether higher dispersal rates in less fragmented landscapes synchronize the dynamics or not. Even if the dynamics synchronize, local population variability can decrease due to indirect effects of dispersal mortality, thereby stabilizing metapopulation dynamics and reducing the risk of extinction. In dynamic landscapes, periodic external perturbations often fully synchronize the dynamics even if they act on a much slower time scale than the ecological interactions. Furthermore, they not only increase the variability of local and metapopulations, but also mostly overrule the effects of habitat fragmentation. This may explain why in nature effects of habitat fragmentation are often small and inconclusive.

ecology↗

Control of ΦC31 integrase-mediated site-specific recombination by protein trans splicing

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system for rapid regulation of site-specific recombination events in vivo. The{varphi} C31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in E. coli generated a fully functional{varphi} C31 integrase. Protein splicing is necessary for recombination activity; no activity was observed when the{varphi} C31 integrase N-and C-terminal extein domains without the intein sequences were co-expressed, nor when a key intein catalytic residue was mutated. As a proof of principle, we used a bistable switch based on an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system. We used araC and tet inducible promoters to regulate the expression of the two parts of the split recombinase. Inversion of a DNA segment containing a constitutive promoter, catalyzed by trans-spliced integrase, switches between RFP and GFP expression only when both inducible promoters are ON. We used the same split inteins to regulate the reconstitution of a split integrase-RDF fusion that efficiently catalyzed the reverse attR x attL recombination, demonstrating that our split-intein regulated recombination system can function as a reversible AND gate in which the forward reaction is catalyzed by the integrase, and the reverse reaction by the integrase-RDF fusion. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

synthetic biology↗

The biggest losers: Habitat isolation deconstructs complex food webs from top to bottom

Habitat fragmentation is threatening global biodiversity. To date, there is only limited understanding of how habitat fragmentation or any alteration to the spatial structure of a landscape in general, affects species diversity within complex ecological networks such as food webs. Here, we present a dynamic and spatially-explicit food web model which integrates complex food web dynamics at the local scale and species-specific dispersal dynamics at the landscape scale, allowing us to study the interplay of local and spatial processes in metacommunities. We explore how habitat fragmentation, defined as a decrease of habitat availability and an increase of habitat isolation, affects the species diversity patterns of complex food webs (-, {beta}-,{gamma} -diversity), and specifically test whether there is a trophic dependency in the effect of habitat fragmentation on species diversity. In our model, habitat isolation is the main driver causing species loss and diversity decline. Our results emphasise that large-bodied consumer species at high trophic positions go extinct faster than smaller species at lower trophic levels, despite being superior dispersers that connect fragmented landscapes better. We attribute the loss of top species to a combined effect of higher biomass loss during dispersal with increasing habitat isolation in general, and the associated energy limitation in highly fragmented landscapes, preventing higher trophic levels to persist. To maintain trophic-complex and species-rich communities calls for effective conservation planning which considers the interdependence of trophic and spatial dynamics as well as the spatial context of a landscape and its energy availability.

ecology↗

Integrating Vectors for Genetic Studies in the Rare Actinomycete Amycolatopsis marina

Few natural product pathways from rare Actinomycetes have been studied due to the difficulty in applying molecular approaches in these genetically intractable organisms. In this study, we sought to identify integrating vectors, derived using phage int/attP loci, that would efficiently integrate site-specifically in the rare Actinomycete, Amycolatopsis marina DSM45569. Analysis of the genome of A. marina DSM45569 indicated the presence of attB-like sequences for TG1 and R4 integrases. The TG1 and R4 attBs were active in in vitro recombination assays with their cognate purified integrases and attP loci. Integrating vectors containing either the TG1 or R4 int/attP loci yielded exconjugants in conjugation assays from E. coli to A. marina DSM45569. Site-specific recombination of the plasmids into the host TG1 or R4 attB sites was confirmed by sequencing. The presence of homologous TG1 and R4 attB sites in other species of this genus indicates that vectors based on TG1 and R4 integrases could be widely applicable.\n\nImportanceRare Actinomycetes have the same potential of natural product discovery as Streptomyces, but the potential has not been fully explored due to the lack of efficient molecular biology tools. In this study, we identified two serine integrases, TG1 and R4, which could be used in the rare Actinomycetes species, Amycolatopsis marina, as tools for genome integration. The high level of conservation between the attB sites for TG1 and R4 in a number of Amycolatopsis species suggested that plasmids with the integration systems from these phages should be widely useful in this genus.

microbiology↗

Somatic inactivating PTPRJ mutations and dysregulated pathways identified in canine melanoma by integrated comparative genomic analysis

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.\n\nAUTHOR SUMMARYMelanoma, an aggressive cancer arising from transformed melanocytes, commonly occurs in pet dogs. Unlike human melanoma, which most often occurs in sun-exposed cutaneous skin, canine melanoma typically arises in sun-shielded oral mucosa. Clinical features of canine melanoma resemble those of human melanoma, particularly the less common sun-shielded human subtypes. However, whereas the genomic basis of diverse human melanoma subtypes is well understood, canine melanoma genomics remain poorly defined. Similarly, although diverse new treatments for human melanoma based on a biologic disease understanding have recently shown dramatic improvements in outcomes for these patients, treatments for canine melanoma are limited and outcomes remain universally poor. Detailing the genomic basis of canine melanoma thus provides untapped potential for improving the lives of pet dogs while also helping to establish canine melanoma as a comparative model system for informing human melanoma biology and treatment. In order to better define the genomic landscape of canine melanoma, we performed multi-platform characterization of 37 tumors. Our integrated analysis confirms that these tumors commonly contain mutations in canine orthologs of human cancer genes such as RAS, MDM2, and TP53 as well mutational patterns that share important similarities with human melanoma subtypes. We have also found a new putative cancer gene, PTPRJ, frequently mutated in canine melanoma. These data will guide additional biologic and therapeutic studies in canine melanoma while framing the utility of comparative studies of canine and human cancers more broadly.

cancer biology↗