Background and aims: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory ...insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need. Methods: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. Results: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. Conclusions: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.
Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact ...the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays.
Demographic aging accompanied by increased falls inevitably leads to an increased incidence of atlantoaxial instabilities (AAI). Minimally invasive surgical procedures decrease the perioperative risk ...and regarding the treatment of AAI, percutaneous transarticular screw fixation of C1/C2 was more frequently considered in the past. This study aims to investigate the outcome of patients treated for AAI by isolated percutaneous transarticular screw fixation of C1/C2 (IPTSFC1/C2) using 3.5 mm fully threaded screws to identify its chances and limitations. In this retrospective study, data from patients who underwent IPTSFC1/C2 were analyzed. 23 patients (17 females and 6 males) with an average age of 73.1 years (y) were included. Mean VAS decreased significantly from preoperative 3.9 ± 1.8 to the last follow-up 2.6 ± 2.5 (p = 0.020) and neurological functions were preserved. In the radiological follow-up, we saw a single malposition of an inserted screw (2.27%) and one single bony fusion (4.54%). However, in 6 of 7 patients (85.71%), there was a loosening of the inserted screws due course. We demonstrated that the use of 3.5 mm fully threaded screws for IPTSFC1/C2 results in low rates of osseous fusions between C1 and C2. Therefore, their use in IPTSFC1/C2 is not suitable, especially for geriatric patients with impaired bone status.
Replenished axial melt lenses at fast-spreading mid-oceanic ridges may move upward and intrude into the overlying hydrothermally altered sheeted dikes, resulting in high-grade contact metamorphism ...with the potential to trigger anatexis in the roof rocks. Assumed products of this process are anatectic melts of felsic composition and granoblastic, two-pyroxene hornfels, representing the residue after partial melting. Integrated Ocean Drilling Program Expeditions 309, 312, and 335 at Site 1256 (eastern equatorial Pacific) sampled such a fossilized oceanic magma chamber. In this study, we simulated magma chamber roof rock anatectic processes by performing partial melting experiments using six different protoliths from the Site 1256 sheeted dike complex, spanning a lithological range from poorly to strongly altered basalts to partially or fully recrystallized granoblastic hornfels. Results show that extensively altered starting material lacking primary magmatic minerals cannot reproduce the chemistry of natural felsic rocks recovered in ridge environments, especially elements sensitive to hydrothermal alteration (e.g., K, Cl). Natural geochemical trends are reproduced through partial melting of moderately altered basalts from the lower sheeted dikes. Two-pyroxene hornfels, the assumed residue, were reproduced only at low melting degrees (<20 vol%). The overall amphibole absence in the experiments confirms the natural observation that amphibole is not produced during peak metamorphism. Comparing experimental products with the natural equivalents reveals that water activity (
a
H
2
O) was significantly reduced during anatectic processes, mainly based on lower melt aluminum oxide and lower plagioclase anorthite content at lower
a
H
2
O. High silica melt at the expected temperature (1000–1050 °C; peak thermal overprint of two-pyroxene hornfels) could only be reproduced in the experimental series performed at
a
H
2
O = 0.1.
At fast-spreading mid-ocean ridges (MORs), the horizon between the axial melt lens (AML) and the overlying sheeted dikes is characterized by extensive anatectic processes. The heat flux of the AML in ...combination with hydrothermal fluids from above causes high-grade contact metamorphism, which may result in anatexis of the roof rocks above the AML. The products of this process are silica-rich anatectic melts that have the potential to contaminate MOR basalts and residual hornfels. Here, we simulate the complex igneous and metamorphic processes occurring at the AML roof by hydrous partial melting experiments and provide corresponding trace element partition coefficients between melt and residues, which are useful to quantify those processes. We present trace element patterns from experimental anatectic felsic melts and the related residue produced by hydrous partial melting of various types of AML roof rocks. The starting materials used are sheeted dikes and hornfelses from Hole 1256D drilled by the Integrated Ocean Drilling Program. Results are compared with directly-related natural lithologies (i.e., felsic veins and granoblastic hornfels) from the same site. The trace element contents generally overlap with natural examples and experimental melts produced at low water activity (aH2O<0.5) can be highly enriched in trace elements despite relatively low SiO2 contents (58.9 to 65.7wt%). A low aH2O is required to reproduce the low Al2O3 contents observed in natural silica-rich rocks. However, low aH2O implies that the presence of residual amphibole is not required for anatectic processes Even though residual amphibole is often used as an important phase for explaining trace element characteristics in relevant felsic rocks formed at MORs when modeling anatexis. Because amphibole is lacking in any experimental residue, which is in agreement with natural hornfelses from the dike/gabbro transition at Site 1256, we assume that partial melting within the AML roof rocks proceeds without the participation of amphibole as residual phase. We present a comprehensive set of trace element compositions as well as bulk and mineral/melt trace element partition coefficients obtained from our amphibole-free experimental results for different potential protoliths over a large range of temperature and at different aH2Os.
The odontoid process (dens) of the second cervical vertebra (axis) is prone to fracture. While the importance of its skeletal integrity has been previously noted, representative three-dimensional ...microarchitecture analyses in humans are not available. This study aimed to determine the bone microarchitecture of the axis using high-resolution quantitative computed tomography (HR-pQCT) and to derive clinical implications for the occurrence and treatment of axis fractures. For initial clinical reference, the apparent density of the axis was determined based on clinical computed tomography (CT) images in patients without and with fractures of the axis. Subsequently, 28 human axes (female 50%) obtained at autopsy were analyzed by HR-pQCT. Analyses were performed in three different regions corresponding to zones I (tip of dens), II (base of dens), and III (corpus axis) of the Anderson and D’Alonzo classification. Lower apparent densities based on clinical CT data were detected in zone II and III compared to zone I in both the group without and with fracture. In the autopsy specimens, cortical thickness and bone volume fraction decreased continuously from zone I to zone III. Trabecular and cortical tissue mineral density was lowest in zone III, with no differences between zones I and II. In conclusion, our clinical and high-resolution ex vivo imaging data highlight a marked regional heterogeneity of bone microarchitecture, with poor cortical and trabecular properties near the dens base. These results may partly explain why zones II and III are at high risk of fracture and osteosynthesis failure.
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