Biology subjects

Laine, J.

Publications and source records attributed to Laine, J..

12 recordsLinked to original sources

bioRxiv2026

Application of the Nicking Loop™ targeted library preparation method to DNBSEQ™ sequencing

Nicking Loop is a PCR-free targeted library preparation method that combines conversion of linear target DNA into circular single-stranded DNA (CssDNA) library with early sample indexing in a single step. The resulting CssDNA libraries can be either directly sequenced or optionally amplified, offering maximum flexibility across sequencing applications. This study demonstrates the compatibility of Nicking Loop circular libraries with a MGIs DNBSEQ platform. Compatibility was evaluated against established linear Nicking Loop libraries sequenced on Illumina MiSeq platform. Using synthetic reference samples with defined variant allele frequencies, Nicking Loop method demonstrated matching performance across both library formats and sequencing platforms. Key quality metrics, including unique molecular identifier (UMI) distributions, error profiles and VAF detection, were all highly consistent. Both library types generated over 97% singleton UMIs, indicating uniform template sampling, and VAF measurements were strongly concordant across platforms (Spearmans {rho} = 0.939). Collectively, these findings demonstrate that Nicking Loop method is directly applicable to circular NGS platforms, such as DNBSEQ, strongly supporting its use as a platform-agnostic library preparation strategy for targeted sequencing applications.

molecular biology↗

bioRxiv2025

Complex patterns of hitchhiking mutation load among stickleback populations

Positive selection causes beneficial alleles to rapidly rise to high frequency in a population. This can cause linked genetic variation to "hitchhike", and thereby also rise in frequency. This linked variation may include deleterious recessive alleles, previously neutrally harbored at low frequency in heterozygous genotypes. Modeling studies have shown that local effective population size and reduced recombination rate should contribute to the probability of deleterious mutations being swept to high frequency by being linked to beneficial alleles in selective sweeps or inversions. Marine sticklebacks have repeatedly adapted to thousands of freshwater habitats that became available after the last ice age, resulting in the formation of distinct marine and freshwater ecotypes. Selection acts on ancient standing genetic variation present in marine populations, causing freshwater-adaptive alleles to increase rapidly over tens of generations. These genomic regions play a key role in the repeated freshwater adaptation of morphological, physiological, and behavioral traits, and evolve under strong selection. Thus, threespine stickleback are an ideal system for investigating the impact of hitchhiking mutation load. We estimate the mutation load in regions of low recombination, including inversions, and the Eda haplotype. We find little evidence for the accumulation of deleterious alleles among genomic regions and populations. Inversions deviated from Hardy-Weinberg equilibrium in several populations, exhibiting an excess of homozygotes. These observations are consistent with purging facilitated by recombination in inversion karyotypes. We conclude that adaptation in threespine stickleback is not limited by hitchhiking deleterious mutations. Significance statementHitchhiking is a ubiquitous force in population genetics, shaping genetic diversity across the genome. Threespine stickleback are a model system for studying parallel adaptation to ecological variation, with low recombination genomic regions known to drive ecological adaptation. A key question is whether a deleterious mutation load is associated with adaptation in this system. We find that hitchhiking mutation load patterns are complex and do not conform to simple model predictions, despite some widespread patterns shared among populations. Our findings suggest that purging in homozygous karyotypes may be critical in reducing deleterious mutation load.

evolutionary biology↗

bioRxiv2025

Nicking Loop™: An infinite amplification of single-stranded circular DNA

Circular single-stranded DNA (CssDNA) offers unique advantages for molecular diagnostics and next-generation sequencing (NGS) due to its nuclease resistance and compatibility with rolling circle amplification. We present the Nicking Loop, a novel and versatile method for converting both single- and double-stranded DNA into CssDNA and enabling robust, unbiased amplification. This technique preserves the original DNA template composition and outperforms PCR in reproducibility and sensitivity, particularly for low variant allele frequencies. We demonstrate that Nicking Loop-amplified CssDNA is directly compatible with NGS platforms utilizing circular DNA as a sequencing template and introduce a novel loop-based indexing strategy that enables efficient sample multiplexing. Furthermore, we confirm the methods compatibility with nanopore sequencing technologies, highlighting its broad applicability across sequencing platforms. Our results establish Nicking Loop as an alternative to conventional linear library preparation, streamlining workflows while enhancing sequencing accuracy and efficiency. Beyond its immediate application in NGS, the method holds promise for broader use in molecular biology, e.g. in DNA data storage. This proof-of-concept study highlights the transformative potential of Nicking Loop in enabling a shift toward circular DNA libraries for new generation of assay preps and precision sequencing applications.

molecular biology↗

bioRxiv2025

Ancient stickleback genomes reveal the chronology of parallel adaptation

Parallel evolution of traits and their underlying genetic basis is well-studied, however, studies of parallel chronology of adaptive genetic changes remain scarce. Threespine stickleback are a model system for studying parallel evolution. We present genomic data from nine subfossil stickleback bones dated to 14.8-0.7 KYR BP in age. Comparing the four highest coverage genomes, which represent different stages along the marine-freshwater continuum, we find that freshwater ancestry is often clustered rather than randomly distributed throughout the marine-freshwater divergent regions of the genome. We consistently find freshwater ancestry on chromosome IV at the early stages of freshwater adaptation. Regions of chromosome IV contain the greatest genetic differentiation between marine and freshwater ecotypes and among the highest density of quantitative trait loci. These include EDA, a large-effect pleiotropic locus associated with defensive armour and variation in neurosensory and behavioural traits. Freshwater ancestry is also found at inversions in the subfossils early in the adaptive process. Our findings add to the growing body of evidence that freshwater adaptation in threespine stickleback has a staggered but predictable temporal dynamic. The probability of parallel genetic change should be increased by the clustering of adaptive alleles in regions of suppressed recombination, particularly for genes that have a large phenotypic effect.

evolutionary biology↗

bioRxiv2024

Alternative splicing of SORBS1 affects neuromuscular junction formation and stability in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular disease characterized by a CTG repeat expansion in the 3" untranslated region of the gene coding for the dystrophia myotonica protein kinase (DMPK). Presence of expanded CTG repeats in DMPK-mRNAs leads to the sequestration of RNA binding factors such as the Muscleblind like (MBNL) proteins resulting in widespread splicing defects contributing to progressive muscle weakness and myotonia. Here, we show that abnormal splicing of SORBS1 exon 25 found in skeletal muscle of myotonic dystrophy type 1 patients is a critical contributor to neuromuscular junction (NMJ) formation and maintenance. Forced exclusion of SORBS1 exon 25 in mice results in NMJ degeneration with marked denervation and postsynaptic destabilization. In zebrafish, misregulation of sorbs1 exon 25 results in reduced motor function and abnormal AChR cluster morphology. Finally, we observed that forcing SORBS1 exon 25 exclusion in hiPSC-derived skeletal muscle cells reduces the formation of large AChR clusters upon agrin stimulation. Thus, our study identifies MBNL regulated SORBS1 alternative splicing during skeletal muscle development as a critical event for NMJ formation and maintenance. The aberrant splicing of SORBS1 exon 25 in DM1 expands our understanding of how splicing dysregulation compromises neuromuscular system communication, shedding light on the broader impact of mRNA splicing regulation on NMJ integrity.

molecular biology↗

bioRxiv2024

Early endosome disturbance and endolysosomal pathway dysfunction in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal dystrophy characterized by the progressive loss of muscle fibers caused by mutations in DMD gene and absence of the dystrophin protein. While autophagy and lysosome biogenesis defects have been described in DMD muscles, the endosomal pathway has never been studied. Here, we showed that impaired lysosome formation is associated with altered acidification and reduced degradative function of the endolysosomal pathway in muscle cells derived from DMD patients. Our data demonstrated that early endosomes are increased in these cells as well as in muscle biopsies from DMD patients and two animal models of DMD, mdx mice and GRMD dogs. We determined that these abnormalities are due to the lack of dystrophin per se and could be correlated with disease progression and severity. We further identified an abnormal upregulation of the Rab5 GTPase protein, one key actor of early endosomal biogenesis and fusion, in the three DMD models which may underlie the endosomal defects. Finally, we demonstrated that Rab5 knock-down in human DMD muscle cells as well as dystrophin restoration in GRMD dogs, normalize Rab5 expression levels and rescue endosomal abnormalities. This study unveils a defect in a pathway essential for muscle homeostasis and for efficacy of adeno associated virus vectors and antisense oligonucleotides-mediated therapies.

pathology↗

bioRxiv2024

Body-wide genetic deficiency of poly(ADP-ribose) polymerase 14 sensitizes mice to colitis

Inflammatory bowel disease (IBD) is a debilitating and relapsing chronic disease of the gastrointestinal tract affecting millions of people. Here, we investigated the expression and functions of poly (ADP-ribose) polymerase 14 (Parp14), an important regulatory protein in immune cells, using a biobank IBD patient cohort as well as two mouse models of colitis, i.e., the IBD-mimicking oral dextran sulfate sodium (DSS) exposure model, and the oral Salmonella exposure model. Parp14 was expressed in the human colon, by cells in the lamina propria, but, in particular, by the epithelial cells with a typical granular staining pattern in the cytosol. The same Parp14 staining pattern was evidenced in both colitis models. Body-wide genetic deficiency of Parp14 in C57BL/6N background sensitized mice to DSS colitis. The Parp14-deficient mice displayed increased rectal bleeding as well as stronger epithelial erosion, Goblet cell loss and immune cell infiltration. The absence of Parp14 did not affect the mouse colon bacterial microbiota based on PacBio long read sequencing. Also, the colon leukocyte populations of Parp14-deficient mice were normal based on flow cytometry. In contrast, we witnessed an altered transcriptional signature in Parp14-deficient mice with bulk tissue RNA-Seq. Gene Ontology (GO)-based classification of differentially expressed genes demonstrated that the colon transcriptional signature of Parp14-deficient mice was dominated by abnormalities in inflammation and infection responses both prior and after the 1-week DSS exposure. Overall, the data indicate that Parp14 has an important role in the maintenance of colon epithelial barrier integrity. The prognostic and predictive biomarker potential of Parp14 in IBD merits further investigation.

immunology↗

bioRxiv2023

The actin-spectrin submembrane scaffold restricts endocytosis along proximal axons

Neuronal clathrin-mediated endocytosis has unique features in compartments such as dendrites and presynaptic boutons, but how membrane and extracellular components are internalized along the axon shaft remains poorly known. Here we focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS), and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. Super-resolution optical microscopy, platinum replica electron microscopy, and their correlative combination on cultured hippocampal neurons reveal that in the AIS, clathrin-coated pits form on bare membrane patches, [~]300 nm circular areas devoid of spectrin mesh and lined by actin filaments we termed "clearings". In fibroblasts and the proximal axon of neurons, spectrin depletion and drug-induced scaffold disorganization increase clathrin-coated pit formation. However, the presence of clathrin-coated pits at the AIS is not directly linked to actual endocytosis: using cargo uptake and live-cell imaging experiments, we find that most AIS clathrin-coated pits are long-lived and immobile within the spectrin mesh clearings. Direct perturbation of the spectrin scaffold as well as elevated neuronal activity could induce endocytosis downstream of clathrin pit formation, showing that spectrin clearings are structures responsible for regulated endocytosis at the AIS.

cell biology↗

bioRxiv2022

Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, caveolae composed of caveolin-3 and Bin1 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Overexpression of Bin1 lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations cause defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1.

cell biology↗

bioRxiv2020

Mutational dynamics and transmission properties of SARS-CoV-2 superspreading events in Austria

Superspreading events shape the COVID-19 pandemic. Here we provide a national-scale analysis of SARS-CoV-2 outbreaks in Austria, a country that played a major role for virus transmission across Europe and beyond. Capitalizing on a national epidemiological surveillance system, we performed deep whole-genome sequencing of virus isolates from 576 samples to cover major Austrian SARS-CoV-2 clusters. Our data chart a map of early viral spreading in Europe, including the path from low-frequency mutations to fixation. Detailed epidemiological surveys enabled us to calculate the effective SARS-CoV-2 population bottlenecks during transmission and unveil time-resolved intra-patient viral quasispecies dynamics. This study demonstrates the power of integrating deep viral genome sequencing and epidemiological data to better understand how SARS-CoV-2 spreads through populations. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2018

Guanabenz treatment improves Oculopharyngeal muscular dystrophy phenotype

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease affecting most profoundly eyelid and pharyngeal muscles, leading respectively to ptosis and dysphagia, and proximal limb muscles at later stages. A short abnormal (GCG) triplet expansion in the polyA- binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. It is commonly accepted that aggregates themselves, the aggregation process and/or the early oligomeric species of PABPN1 are toxic in OPMD. Decreasing PABPN1 aggregate load in animal models of OPMD ameliorates the muscle phenotype. In order to identify a potential therapeutic molecule that would prevent and reduce aggregates, we tested guanabenz acetate (GA), an FDA-approved antihypertensive drug, in OPMD cells as well as in the A17 OPMD mouse model. We demonstrate that treating mice with GA reduces the size and number of nuclear aggregates, improves muscle force, protects myofibres from the pathology-derived turnover and decreases fibrosis. GA is known to target various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. Here we used a cellular model of OPMD to demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiator factor 2 subunit (eIF2) and the splicing of Xbp1, key components of the UPR. Altogether these data suggest that modulation of protein folding regulation can be beneficial for OPMD and support the further development of guanabenz or its derivatives for treatment of OPMD in humans.\n\nSignificance StatementOculopharyngeal muscular dystrophy (OPMD) is a rare late onset incurable genetic disease characterized by the formation of insoluble aggregates in skeletal muscles. It has been shown that the reduction of aggregates correlates with an improvement of the disease. Here we used a mouse model of OPMD to show that Guanabenz acetate, the active constituent of a marketed but recently discontinued drug for hypertension, decreases the number and the size of aggregates after systemic delivery and improves many aspects of the disease. We also describe experimental evidences explaining the mechanism behind the efficacy of such compound for OPMD.

pathology↗

bioRxiv2018

Clathrin plaques form mechanotransducing platforms

Large flat clathrin plaques are stable features of the plasma membrane associated with sites of strong adhesion suggesting that they could also play a role in force transduction. Here, we analyzed how clathrin plaques interact with the cytoskeleton and how they respond to mechanical cues in skeletal muscle myotubes. We show that branched actin networks surrounding clathrin plaques are directly regulated by dynamin 2, anchor intermediate filaments and sequester YAP at the plasma membrane. Dynamin 2, clathrin and desmin intermediate filaments are all required for basal YAP nucleocytoplasmic distribution and efficient nuclear translocation in response to mechanical stimuli. Dynamin 2 mutations that are responsible for centronuclear myopathy in humans disorganize the desmin network and deregulate YAP signaling both in vitro and in vivo. Thus, clathrin plaques and associated dynamin 2 are defined here as a new sensor conveying mechanical cues and integrate cell signaling with cytoskeletal regulation.

cell biology↗