Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD ...patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of ‘structural weakness’, intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah−/− diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah−/− diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah−/− single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah−/− genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.
Ventilator-induced diaphragm dysfunction (VIDD) is a common sequela of intensive care unit (ICU) treatment requiring mechanical ventilation (MV) and neuromuscular blockade (NMBA). It is characterised ...by diaphragm weakness, prolonged respirator weaning and adverse outcomes. Dissociative glucocorticoids (e.g., vamorolone, VBP-15) and chaperone co-inducers (e.g., BGP-15) previously showed positive effects in an ICU-rat model. In limb muscle critical illness myopathy, preferential myosin loss prevails, while myofibrillar protein post-translational modifications are more dominant in VIDD. It is not known whether the marked decline in specific force (force normalised to cross-sectional area) is a pure consequence of altered contractility signaling or whether diaphragm weakness also has a structural correlate through sterical remodeling of myofibrillar cytoarchitecture, how quickly it develops, and to which extent VBP-15 or BGP-15 may specifically recover myofibrillar geometry. To address these questions, we performed label-free multiphoton Second Harmonic Generation (SHG) imaging followed by quantitative morphometry in single diaphragm muscle fibres from healthy rats subjected to five or 10 days of MV + NMBA to simulate ICU treatment without underlying confounding pathology (like sepsis). Rats received daily treatment of either Prednisolone, VBP-15, BGP-15 or none. Myosin-II SHG signal intensities, fibre diameters (FD) as well as the parameters of myofibrillar angular parallelism (cosine angle sum, CAS) and in-register of adjacent myofibrils (Vernier density, VD) were computed from SHG images. ICU treatment caused a decline in FD at day 10 as well as a significant decline in CAS and VD from day 5. Vamorolone effectively recovered FD at day 10, while BGP-15 was more effective at day 5. BGP-15 was more effective than VBP-15 in recovering CAS at day 10 although not to control levels. In-register VD levels were restored at day 10 by both compounds. Our study is the first to provide quantitative insights into VIDD-related myofibrillar remodeling unravelled by SHG imaging, suggesting that both VBP-15 and BGP-15 can effectively ameliorate the structure-related dysfunction in VIDD.
Abstract
Background
A common polymorphism (R577X) in the
ACTN3
gene results in the complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~ 16% of the world’s ...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
L
0
+ 20% stretch did not cause eccentric damage. In contrast,
L
0
+ 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.
Methods
Single 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.
Results
We show that the absence of α-actinin-3 does not affect the visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned
Actn3KO
fibres are mechanically weaker being prone to breakage when eccentrically stretched. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of
Actn3KO
fast-twitch fibres with an increase in Y-shaped myofibrillar branching.
Conclusions
The absence of α-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
L
0
+ 30%, but not
L
0
+ 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-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 α-actinin-3 deficient fibres will be increased, resulting in fibre breakages.
Deep learning (DL) shows notable success in biomedical studies. However, most DL algorithms work as black boxes, exclude biomedical experts, and need extensive data. This is especially problematic ...for fundamental research in the laboratory, where often only small and sparse data are available and the objective is knowledge discovery rather than automation. Furthermore, basic research is usually hypothesis-driven and extensive prior knowledge (priors) exists. To address this, the Self-Enhancing Multi-Photon Artificial Intelligence (SEMPAI) that is designed for multiphoton microscopy (MPM)-based laboratory research is presented. It utilizes meta-learning to optimize prior (and hypothesis) integration, data representation, and neural network architecture simultaneously. By this, the method allows hypothesis testing with DL and provides interpretable feedback about the origin of biological information in 3D images. SEMPAI performs multi-task learning of several related tasks to enable prediction for small datasets. SEMPAI is applied on an extensive MPM database of single muscle fibers from a decade of experiments, resulting in the largest joint analysis of pathologies and function for single muscle fibers to date. It outperforms state-of-the-art biomarkers in six of seven prediction tasks, including those with scarce data. SEMPAI's DL models with integrated priors are superior to those without priors and to prior-only approaches.
A quality assurance/quality control program was implemented in the framework of the EU project HBM4EU to assess and improve the comparability of biomarker analysis and to build a network of competent ...laboratories. Four rounds of proficiency tests were organized for 15 phthalate and two DINCH urinary biomarkers (0.2-138 ng/mL) over a period of 18 months, with the involvement of 28 laboratories. A substantial improvement in performance was observed after the first round in particular, and by the end of the program, an average satisfactory performance rate of 90% was achieved. The interlaboratory reproducibility as derived from the participants' results varied for the various biomarkers and rounds, with an average of 24% for the biomarkers of eight single-isomer phthalates (e.g., DnBP and DEHP) and 43% for the more challenging biomarkers of the mixed-isomer phthalates (DiNP, DiDP) and DINCH. When the reproducibility was based only on the laboratories that consistently achieved a satisfactory performance, this improved to 17% and 26%, respectively, clearly demonstrating the success of the QA/QC efforts. The program thus aided in building capacity and the establishment of a network of competent laboratories able to generate comparable and accurate HBM data for phthalate and DINCH biomarkers in 14 EU countries. In addition, global comparability was ensured by including external expert laboratories.
More than 20 years ago, acrylamide was added to the list of potential carcinogens found in many common dietary products and tobacco smoke. Consequently, human biomonitoring studies investigating ...exposure to acrylamide in the form of adducts in blood and metabolites in urine have been performed to obtain data on the actual burden in different populations of the world and in Europe. Recognizing the related health risk, the European Commission responded with measures to curb the acrylamide content in food products. In 2017, a trans-European human biomonitoring project (HBM4EU) was started with the aim to investigate exposure to several chemicals, including acrylamide. Here we set out to provide a combined analysis of previous and current European acrylamide biomonitoring study results by harmonizing and integrating different data sources, including HBM4EU aligned studies, with the aim to resolve overall and current time trends of acrylamide exposure in Europe. Data from 10 European countries were included in the analysis, comprising more than 5500 individual samples (3214 children and teenagers, 2293 adults). We utilized linear models as well as a non-linear fit and breakpoint analysis to investigate trends in temporal acrylamide exposure as well as descriptive statistics and statistical tests to validate findings. Our results indicate an overall increase in acrylamide exposure between the years 2001 and 2017. Studies with samples collected after 2018 focusing on adults do not indicate increasing exposure but show declining values. Regional differences appear to affect absolute values, but not the overall time-trend of exposure. As benchmark levels for acrylamide content in food have been adopted in Europe in 2018, our results may imply the effects of these measures, but only indicated for adults, as corresponding data are still missing for children.
Acrylamide, a substance potentially carcinogenic in humans, represents a very prevalent contaminant in food and is also contained in tobacco smoke. Occupational exposure to higher concentrations of ...acrylamide was shown to induce neurotoxicity in humans. To minimize related risks for public health, it is vital to obtain data on the actual level of exposure in differently affected segments of the population. To achieve this aim, acrylamide has been added to the list of substances of concern to be investigated in the HBM4EU project, a European initiative to obtain biomonitoring data for a number of pollutants highly relevant for public health. This report summarizes the results obtained for acrylamide, with a focus on time-trends and recent exposure levels, obtained by HBM4EU as well as by associated studies in a total of seven European countries. Mean biomarker levels were compared by sampling year and time-trends were analyzed using linear regression models and an adequate statistical test. An increasing trend of acrylamide biomarker concentrations was found in children for the years 2014–2017, while in adults an overall increase in exposure was found to be not significant for the time period of observation (2000–2021). For smokers, represented by two studies and sampling for, over a total three years, no clear tendency was observed. In conclusion, samples from European countries indicate that average acrylamide exposure still exceeds suggested benchmark levels and may be of specific concern in children. More research is required to confirm trends of declining values observed in most recent years.
The architectural structure of cells is essential for the cells' function, which becomes especially apparent in the highly "structure functionally" tuned skeletal muscle cells. Here, structural ...changes in the microstructure can have a direct impact on performance parameters, such as isometric or tetanic force production. The microarchitecture of the actin-myosin lattice in muscle cells can be detected noninvasively in living cells and in 3D by using second harmonic generation (SHG) microscopy, forgoing the need to alter samples by introducing fluorescent probes into them. Here, we provide tools and step-by-step protocols to guide the processes of obtaining SHG microscopy image data from samples, as well as extracting characteristic values from the image data to quantify the cellular microarchitecture using characteristic patterns of myofibrillar lattice alignments.
Abstract Introduction Biomonitoring is the most powerful approach to assess individual exposure and health risk of workers from chemical substances. However, availability and implementation of ...published biomonitoring methods often pose notable challenges. In 1955 the Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) was founded by the Deutsche Forschungsgemeinschaft. Within the Commission, the working group “Analyses of Hazardous Substances in Biological Materials” (AiBM) develops, verifies, and publishes procedures for the determination of biomonitoring parameters. Methods AiBM applies a multi-stage process to develop and evaluate biomonitoring methods. Within this process it is essential that every method is verified by a second laboratory to ensure the reproducibility of the analytical procedure and the reliability data. Submitted methods and examination reports are discussed within the working group. Positively evaluated methods are adopted for publication. Others are returned to the developer for revision. Methods with fundamental drawbacks are rejected. Adopted methods were published first in a book series and, since 2016, in the quarterly online journal “The MAK Collection for Occupational Health and Safety”. Results and Discussion Since 1985, AibM has published nearly 200 analytical methods in English. Such methods include inorganic parameters (e.g. Pb, Hg, As) as well as organic parameters (e.g. solvents, plasticizers, pesticides) and are available online free of charge (https://onlinelibrary.wiley.com/doi/book/10.1002/3527600418 and https://series.publisso.de/de/pgseries/overview/mak/dam). Conclusion These detailed, ready-to-use protocols for human biomonitoring enable the monitoring of occupational exposure and often the determination of the background exposure in the general population as well.
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