The verbal class which is characterized by a suffix -nu- in the infinitive and -ne- in the present shows considerable dialectal diversity in Croatian and in the other western South Slavic languages... Show moreThe verbal class which is characterized by a suffix -nu- in the infinitive and -ne- in the present shows considerable dialectal diversity in Croatian and in the other western South Slavic languages. In many dialects, verbs belonging to this class have a suffix -ni- in the infinitive, aorist, participles and/or present. The aim of this paper is to explain how these dialectal forms and the relevant dialectal isoglosses have arisen. It argues that the allomorph -ni- < *-nywas originally found in the infinitive, while -nu- < *-nǫ- used to be restricted to the aorist. Show less
Eggelbusch, M.; Shi, A.D.; Broeksma, B.C.; Vazquez-Cruz, M.; Soares, M.N.; Wit, G.M.J. de; ... ; Wust, R.C.I. 2022
Background Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain... Show moreBackground Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement.Methods Differentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100-200 ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3-induced IL-1 ss production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function.Results NLRP3 gene expression and protein concentrations increased in a time-dependent and dose-dependent manner. Intracellular IL-1 ss concentration significantly increased (P < 0.0001), but significantly less with MCC950 (P = 0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24 h (P = 0.03), and myotubes remained smaller up to 72 h (P = 0.0009). Exposure of myotubes to IL-1 ss caused similar alterations in cell morphology, and MCC950 mitigated these LPS-induced differences in cell diameter. NLRP3 appeared to co-localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P = 0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation.Conclusions LPS- induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association. Show less
Stel, W. van der; Yang, H.; Le Dévédec, S.E.; Water, B. van de; Beltman, J.B.; Danen, E.H.J. 2022
Cells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation,... Show moreCells can adjust their mitochondrial morphology by altering the balance between mitochondrial fission and fusion to adapt to stressful conditions. The connection between a chemical perturbation, changes in mitochondrial function, and altered mitochondrial morphology is not well understood. Here, we made use of high-throughput high-content confocal microscopy to assess the effects of distinct classes of oxidative phosphorylation (OXPHOS) complex inhibitors on mitochondrial parameters in a concentration and time resolved manner. Mitochondrial morphology phenotypes were clustered based on machine learning algorithms and mitochondrial integrity patterns were mapped. In parallel, changes in mitochondrial membrane potential (MMP), mitochondrial and cellular ATP levels, and viability were microscopically assessed. We found that inhibition of MMP, mitochondrial ATP production, and oxygen consumption rate (OCR) using sublethal concentrations of complex I and III inhibitors did not trigger mitochondrial fragmentation. Instead, complex V inhibitors that suppressed ATP and OCR but increased MMP provoked a more fragmented mitochondrial morphology. In agreement, complex V but not complex I or III inhibitors triggered proteolytic cleavage of the mitochondrial fusion protein, OPA1. The relation between increased MMP and fragmentation did not extend beyond OXPHOS complex inhibitors: increasing MMP by blocking the mPTP pore did not lead to OPA1 cleavage or mitochondrial fragmentation and the OXPHOS uncoupler FCCP was associated with OPA1 cleavage and MMP reduction. Altogether, our findings connect vital mitochondrial functions and phenotypes in a high-throughput high-content confocal microscopy approach that help understanding of chemical-induced toxicity caused by OXPHOS complex perturbing chemicals. Show less
