3D-surface reconstruction of cellular cryo-soft X-ray microscopy tomograms using semi-supervised deep learning

Cryo-soft X-ray tomography (cryo-SXT) is a powerful method to investigate the ultrastructure of cells, offering resolution in the tens of nm range and strong contrast for membranous structures without requirement for labeling or chemical fixation. The short acquisition time and the relatively large volumes acquired allow for fast acquisition of large amounts of tomographic image data. Segmentation of these data into accessible features is a necessary step in gaining biologically relevant information from cryo-soft X-ray tomograms. However, manual image segmentation still requires several orders of magnitude more time than data acquisition. To address this challenge, we have here developed an end-to-end automated 3D-segmentation pipeline based on semi-supervised deep learning. Our approach is suitable for high-throughput analysis of large amounts of tomographic data, while being robust when faced with limited manual annotations and variations in the tomographic conditions. We validate our approach by extracting three-dimensional information on cellular ultrastructure and by quantifying nanoscopic morphological parameters of filopodia in mammalian cells

Heart failure after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous DES-p.R349P knock-in mice

Background Mutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p. R350P. Methods and results Transverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 ± 2.9%, HET-sham: 64.5 ± 4.7%, WTTAC: 63.5 ± 4.9%, HET-TAC: 55.7 ± 5.4%; p<0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 ± 2385 μl/min, HET sham: 10391 ± 1349μl/min, WT-TAC: 8097 ± 1903μl/min, HET-TAC: 5793 ± 2517μl/min; p<0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 ± 406, HET-sham: 13526 ± 781, WT-TAC: 11155 ± 3315, HET-TAC: 8649 ± 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC. Conclusion Pressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure

Datasets for training, validation and hyperparameter optimization of the deep network

Cryo-soft X-ray tomography (cryo-SXT) is a powerful method to investigate the ultrastructure of cells, offering resolution in the tens of nm range and strong contrast for membranous structures without requirement for labeling or chemical fixation. The short acquisition time and the relatively large volumes acquired allow for fast acquisition of large amounts of tomographic image data. Segmentation of these data into accessible features is a necessary step in gaining biologically relevant information from cryo-soft X-ray tomograms. However, manual image segmentation still requires several orders of magnitude more time than data acquisition. To address this challenge, we have here developed an end-to-end automated 3D-segmentation pipeline based on semi-supervised deep learning. Our approach is suitable for high-throughput analysis of large amounts of tomographic data, while being robust when faced with limited manual annotations and variations in the tomographic conditions. We validate our approach by extracting three-dimensional information on cellular ultrastructure and by quantifying nanoscopic morphological parameters of filopodia in mammalian cells

Malignant cardiac phenotype after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous R349P desmin knock-in mice

Mutations of the human desmin gene on chromosome 2q35 cause autosomal-dominant and -recessive myopathies and ardiomyopathies. For pathophysiological and therapeutic studies, we generated a R349P desmin knock-in mouse strain, which serves as patient-mimicking disease model for desminopathies. Here, we investigated the influence of pressure overload on cardiac function in heterozygous R349P desmin knock-in mice. Young, non-operated heterozygous R349P desmin knock-in mice do not show obvious signs of cardiomyopathy. When subjected to pressure overload by TAC operation, these mice develop marked signs of cardiomyopathy with accompanying arrhythmias and atrioventricular blocks. The cardiac disease manifestations are associated with reductions in mitochondrial respiratory chain complex activities and mitochondrial DNA copy numbers. Preventive avoidance of pressure overload, i.e. arterial hypertension, to the heart may thus represent a crucial therapeutic measure. Heterozygous R349P desmin knock-in mice and their wild-type siblings were used. Transverse aortic constriction (TAC) operation was performed to induce pressure overload. Cardiac function was analyzed by left heart catheterization, electrophysiological investigation, and echocardiography. Moreover, mitochondrial respiratory complex activities, mtDNA copy number, and mtDNA integrity were investigated

The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex

The increasing prevalence of multidrug-resistant pathogenic bacteria is making current antibiotics obsolete. Proline-rich antimicrobial peptides (PrAMPs) display potent activity against Gram-negative bacteria and thus represent an avenue for antibiotic development. PrAMPs from the oncocin family interact with the ribosome to inhibit translation, but their mode of action has remained unclear. Here we have determined a structure of the Onc112 peptide in complex with the Thermus thermophilus 70S ribosome at a resolution of 3.1 angstrom by X-ray crystallography. The Onc112 peptide binds within the ribosomal exit tunnel and extends toward the peptidyl transferase center, where it overlaps with the binding site for an aminoacyl-tRNA. We show biochemically that the binding of Onc112 blocks and destabilizes the initiation complex, thus preventing entry into the elongation phase. Our findings provide a basis for the future development of this class of potent antimicrobial agents