Biology subjects

Haug, M.

Publications and source records attributed to Haug, M..

9 recordsLinked to original sources

bioRxiv2022

Effect of insulin insufficiency on ultrastructure and function in skeletal muscle

BackgroundDecreased insulin availability and high blood glucose levels, the hallmark features of poorly controlled diabetes, drive disease progression and are associated with decreased skeletal muscle mass. We have shown that mice with {beta}-cell dysfunction and normal insulin sensitivity have decreased skeletal muscle mass. This project asks how insulin deficiency impacts on the structure and function of the remaining skeletal muscle in these animals. MethodsSkeletal muscle function was determined by measuring exercise capacity and specific muscle strength prior to and after insulin supplementation for 28 days in 12-week-old mice with conditional {beta}-cell deletion of the ATP binding cassette transporters ABCA1 and ABCG1 ({beta}-DKO mice). Abca1 and Abcg1 floxed (fl/fl) mice were used as controls. RNAseq was used to quantify changes in transcripts in soleus and extensor digitorum longus muscles. Skeletal muscle and mitochondrial morphology were assessed by transmission electron microscopy. Myofibrillar Ca2+ sensitivity and maximum isometric single muscle fibre force were assessed using MyoRobot biomechatronics technology. ResultsRNA transcripts were significantly altered in {beta}-DKO mice compared to fl/fl controls (32 in extensor digitorum longus and 412 in soleus). Exercise capacity and muscle strength were significantly decreased in {beta}-DKO mice compared to fl/fl controls (p = 0.012), and a loss of structural integrity was also observed in skeletal muscle from the {beta}-DKO mice. Supplementation of {beta}-DKO mice with insulin restored muscle integrity, strength and expression of 13 and 16 of the dysregulated transcripts in and extensor digitorum longus and soleus muscles, respectively. ConclusionsInsulin insufficiency due to {beta}-cell dysfunction perturbs the structure and function of skeletal muscle. These adverse effects are rectified by insulin supplementation.

physiology↗

bioRxiv2022

SEMPAI: a Self-Enhancing Multi-Photon Artificial Intelligence for prior-informed assessment of muscle function and pathology

Deep learning (DL) shows notable success in biomedical studies. However, most DL algorithms work as a black box, exclude biomedical experts, and need extensive data. We introduce the Self-Enhancing Multi-Photon Artificial Intelligence (SEMPAI), that integrates hypothesis-driven priors in a data-driven DL approach for research on multiphoton microscopy (MPM) of muscle fibers. SEMPAI utilizes meta-learning to optimize prior integration, data representation, and neural network architecture simultaneously. This allows hypothesis testing and provides interpretable feedback about the origin of biological information in MPM images. SEMPAI performs joint learning of several tasks to enable prediction for small datasets. The method is applied on an extensive multi-study dataset resulting in the largest joint analysis of pathologies and function for single muscle fibers. SEMPAI outperforms state-of-the-art biomarkers in six of seven predictive tasks, including those with scarce data. SEMPAIs DL models with integrated priors are superior to those without priors and to prior-only machine learning approaches.

bioinformatics↗

bioRxiv2021

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics

BackgroundA common polymorphism (R577X) in the ACTN3 gene results in complete absence of the Z-disc protein -actinin-3 from fast-twitch muscle fibres in ~16% of the worlds population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0+ 20% stretch did not cause eccentric damage. In contrast, L0+ 30% stretch produced a significant ~40% deficit in maximum force; here we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this. MethodsSingle fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means {+/-} SD and tested for significance using ANOVA. ResultsWe show that the absence of -actinin-3 does not affect the unloaded maximum speed of contraction, visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically contracted. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar lattice shifts. ConclusionsAbsence of -actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that, in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0+ 30%, but not, L0+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why -actinin-3 deficient humans have been reported to have a faster decline in muscle function with increasing age, that is; as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional -actinin-3 deficient fibres will be increased, resulting in fibres breakages.

physiology↗

bioRxiv2020

The proteomic landscape of resting and activated CD4+ T cells reveal insights into cell differentiation and function

CD4+ T cells (T helper cells) are cytokine-producing adaptive immune cells that activate or regulate the responses of various immune cells. The activation and functional status of CD4+ T cells is important for adequate responses to pathogen infections but has also been associated with auto-immune disorders and survival in several cancers. In the current study, we carried out a label-free high-resolution FTMS-based proteomic profiling of resting and T cell receptor-activated (72h) primary human CD4+ T cells from peripheral blood of healthy donors as well as SUP-T1 cells. We identified 5,237 proteins, of which significant alterations in the levels of 1,119 proteins were observed between resting and activated CD4+ T cells. We confirmed several known T-cell activation-related processes such as IL-2 response, metabolic and signaling changes, cell cycle induction, differentiation into effector cells among others. Several stimulatory/inhibitory immune checkpoint markers were altered considerably between resting and activated CD4+ T cells. Network analysis identified several known regulatory hubs of CD4+ T cell activation, including IFNG, IRF1, FOXP3, AURKA, and novel hubs such as RIOK2. Comparison of primary CD4+ T cell proteomic profiles with human lymphoblastic cell lines revealed a substantial overlap, while comparison with mouse CD+ T cell data suggested interspecies proteomic differences.

immunology↗

bioRxiv2020

Biomarker signatures of quality for nasal chondrocyte-derived engineered cartilage

The definition of quality controls for cell therapy and engineered product manufacturing processes is critical for safe, effective, and standardized clinical implementation. Using the example context of cartilage grafts engineered from autologous nasal chondrocytes, currently used for articular cartilage repair in a phase II clinical trial, we outlined how gene expression patterns and generalized linear models can be introduced to define molecular signatures of identity, purity, and potency. We first verified that cells from the biopsied nasal cartilage can be contaminated by cells from a neighboring tissue, namely perichondrial cells, and discovered that they cannot deposit cartilaginous matrix. Differential analysis of gene expression enabled the definition of identity markers for the two cell populations, which were predictive of purity in mixed cultures. Specific patterns of expression of the same genes were significantly correlated with cell potency, defined as the capacity to generate tissues with histological and biochemical features of hyaline cartilage. The outlined approach can now be considered for implementation in a Good Manufacturing Practice setting, and offers a paradigm for other regenerative cellular therapies.

bioengineering↗

bioRxiv2019

Growing old too early, automated assessment of skeletal muscle single fiber biomechanics in ageing R349P desmin knock-in mice using the MyoRobot technology

Muscle biomechanics is determined by active motor-protein assembly and passive strain transmission through cytoskeletal structures. The extrasarcomeric desmin filament network aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness compatible with a pre-aged phenotype in the disease. Since the specific effect of R349P desmin on axial biomechanics in fully differentiated muscle fibers is unknown, we used our automated MyoRobot biomechatronics platform to compare passive and active biomechanics in single fibers derived from fast- and slow-twitch muscles from adult to senile mice hetero- or homozygous for this desmin mutation with wild-type littermates. Experimental protocols involved caffeine-induced Ca2+-mediated force transients, pCa-force curves, resting length-tension curves, visco-elasticity and slack-tests. We demonstrate that the presence of R349P desmin predominantly increased single fiber axial stiffness in both muscle types with a pre-aged phenotype over wild-type fibers. Axial viscosity was unaffected. Likewise, no systematic changes in Ca2+-mediated force properties were found. Notably, mutant single fibers showed faster unloaded shortening over wild-type fibers. Effects of ageing seen in the wild-type always appeared earlier in the mutant desmin fibers. Impaired R349P desmin muscle biomechanics is clearly an effect of a compromised intermediate filament network rather than secondary to fibrosis.

physiology↗

bioRxiv2019

Global assessment of Mycobacterium avium subspecies hominissuis genetic requirement for growth and virulence.

Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and in vitro growth, and organized ~3500 identified transposon mutants for hypothesis-driven research. The majority (71 %) of the genes we identified as essential for MAH in vitro had a growth-essential mutual ortholog in the related and highly virulent M. tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. Strikingly, > 97 % of the MAH virulence genes had a mutual ortholog in Mtb. Two of the three virulence genes specific to MAH (i.e. no Mtb mutual orthologs) were PPE proteins, a family of proteins unique to mycobacteria and highly associated with virulence. Finally, we validated novel genes as required for successful MAH infection; one encoding a probable MFS transporter and another a hypothetical protein located in immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection.\n\nAuthor summaryPulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the M. avium complex (MAC), whereof >90% arise from Mycobacterium avium subsp. hominissuis (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12 months. To control MAH by improved therapy, prevention and diagnostics, we need to understand the underlying mechanisms of infection. While genetic manipulation of pathogens is crucial to study pathogenesis, M. avium (Mav) has been found notoriously hard to engineer. Here, we identify an MAH strain highly susceptible to high-density transposon mutagenesis and transformation, facilitating genetic engineering and analysis of gene function. We provide crucial insights into this strains global genetic requirements for growth and infection. Surprisingly, we find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M. tuberculosis (Mtb). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment, or translate to broader treatment of related mycobacterial diseases.

microbiology↗

bioRxiv2018

Multi-omics approach identifies novel pathogen-derived prognostic biomarkers in patients with Pseudomonas aeruginosa bloodstream infection

Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.

microbiology↗

bioRxiv2018

Genetic variation/evolution and differential host responses resulting from in-patient adaptation of Mycobacterium avium

Mycobacterium avium (Mav) complex (MAC) are characterized as non-tuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact on the infection outcome. The current study aimed to characterize the genomic changes within Mav isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed field gel electrophoresis and whole genome sequencing was performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Patients (4, 9 and 13) who contributed more than two isolates were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single nucleotide polymorphisms (SNPs), suggesting that Mav accumulates mutations at high rates during persistent infections. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency towards increased persistence and down-regulation of inflammatory cytokines by host-adapted Mav strains. The study revealed rapid genetic evolution of Mav in chronically infected patients accompanied with change in virulence properties of the sequential mycobacterial isolates.\n\nIMPORTANCEMAC are a group of opportunistic pathogens, consisting of Mav and M. intracellulare species. Mav is found ubiquitously in the environment. In Mav infected individuals, Mav has been known to persist for long periods of time, and anti-mycobacterial drugs are unable to effectively clear the infection. The continued presence of the bacteria, could be attributed to either a single persistent strain or reinfection with the same or different strain. We examined sequential isolates collected over time from Mav infected individuals and observed that most patients carried the same strain overtime and were not re infected. We observed high rates of mutation within the serial isolates, accompanied with changes in virulence properties. In the light of increase in incidence of MAC related infections, this study highlights the possibility that host adapted Mav undergo genetic modifications to cope with the host environment and thereby persisting longer.

immunology↗