An improved method for high-throughput quantification of autophagy in mammalian cells

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

Padma Kant Shukla July 7, 1950 - January 26, 2013

Professor Padma Kant Shukla passed away suddenly on 26 January 2013 during his travel to New Delhi, India, the day after receiving the prestigious Hind Rattan (Jewel of India) Award. He was born 7 July 1950 in the village Tulapur, Uttar Pradesh (UP), India and was educated there. He graduated with Ph.D. in Physics from Banaras Hindu University in Varanasi, India at age 22. In 1975, he obtained his second doctorate degree in Theoretical Plasma Physics from Umea University under the supervision of one of us (L.S.). Affiliated with 14 academic and national institutions around the world throughout his career, his primary affiliation for 40 years was the Faculty of Physics & Astronomy, Ruhr-University Bochum (RUB), Germany, where in July 2010 he was appointed RUB International Chair, a lifetime Distinguished Physics Professor position. Padma Shukla published about 1500 papers in prestigious journals, playing very often a leading role in the exploration of new frontiers of plasma science, with special emphasis on the physics of dusty plasmas, on neutrino plasma physics, on nonlinear wave phenomena including parametric instabilities, on solitons and vortices, and on dense quantum plasma modes and structures. He was passionate about science and had an infectious enthusiasm that touched everyone who knew him

The Hamburg Spondylodiscitis Assessment Score (HSAS) for Immediate Evaluation of Mortality Risk on Hospital Admission

(1) Background: Patients with spondylodiscitis often present with unspecific and heterogeneous symptoms that delay diagnosis and inevitable therapeutic steps leading to increased mortality rates of up to 27%. A rapid initial triage is essential to identify patients at risk for a complicative disease course. We therefore aimed to develop a risk assessment score using fast available parameters to predict in-hospital mortality of patients admitted with spondylodiscitis. (2) Methods: A retrospective data analysis of 307 patients with spondylodiscitis recruited from 2013 to 2020 was carried out. Patients were grouped according to all-cause mortality. Via logistic regression, individual patient and clinical characteristics predictive of mortality were identified. A weighted sum score to estimate a patient’s risk of mortality was developed and validated in a randomly selected subgroup of spondylodiscitis patients. (3) Results: 14% of patients with spondylodiscitis died during their in-hospital stay at a tertiary center for spinal surgery. Univariate and logistic regression analyses of parameters recorded at hospital admission showed that age older than 72.5 years, rheumatoid arthritis, creatinine > 1.29 mg/dL and CRP > 140.5 mg/L increased the risk of mortality 3.9-fold, 9.4-fold, 4.3-fold and 4.1-fold, respectively. S. aureus detection increased the risk of mortality by 2.3-fold. (4) Conclusions: The novel Hamburg Spondylodiscitis Assessment Score (HSAS) shows a good fit identifying patients at low-, moderate-, high- and very high risk for in hospital mortality on admission (AUC: 0.795; p < 0.001). The implementation of the HSAS into clinical practice could ease identification of high-risk patients using readily available parameters alone, improving the patient’s safety and outcome

Anatexis at the roof of an oceanic magma chamber at IODP Site 1256 (equatorial Pacific): an experimental study

International audienceReplenished 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 (aH2O) was significantly reduced during anatectic processes, mainly based on lower melt aluminum oxide and lower plagioclase anorthite content at lower aH2O. 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 aH2O = 0.1

Outcome after Posterior Vertebral Column Resection in Patients with Severe Osteoporotic Fractures&mdash;A Retrospective Analysis from Two Centers

Background and Objectives: In osteoporotic fractures of the spine with resulting kyphosis and threatening compression of neural structures, therapeutic decisions are difficult. The posterior vertebral column resection (pVCR) has been described by different authors as a surgical treatment in a single-stage posterior procedure. The aim of this study is to evaluate midterm outcomes of patients treated by pVCR due to severe osteoporotic fractures. Materials and Methods: Retrospective data analysis of all the patients treated for osteoporotic fractures by pVCR from 2012&ndash;2020 at two centers was performed. Demographic data, visual analog scale (VAS), Frankel scale (FS), Karnofsky performance status (KPS), radiological result and spinal fusion rates were evaluated. Results: A total of 17 patients were included. The mean age was 70 &plusmn; 10.2 y. The mean VAS decreased significantly from 7.7 &plusmn; 2.8 preoperatively to 3.0 &plusmn; 1.6 at last follow-up (p &lt; 0.001) and the segmental kyphosis decreased from 29.4 &plusmn; 14.1&deg; to 7.9 &plusmn; 8.0&deg; (p &lt; 0.001). The neurologic function on the FS did not worsen in any and improved in four of the patients. The median KPS remained stable over the whole observation period (70% vs. 70%). Spinal fusion was observed in nine out of nine patients who received CT follow-up &gt;120 days after index surgery. Conclusions: This study showed that pVCR is a safe surgical technique with few surgical complications and no neurological deterioration considering the cohort. The patients&rsquo; segmental kyphosis and VAS improved significantly, while the KPS remained stable

Spontaneous spondylodiscitis and endocarditis: interdisciplinary experience from a tertiary institutional case series and proposal of a treatment algorithm

Previously, the simultaneous presence of endocarditis (IE) has been reported in 3-30% of spondylodiscitis cases. The specific implications on therapy and outcome of a simultaneous presence of both diseases are not yet fully evaluated. Therefore, the aim of this study was to investigate the influence of a simultaneously present endocarditis on the course of therapy and outcome of spondylodiscitis. A prospective database analysis of 328 patients diagnosed with spontaneous spondylodiscitis (S) using statistical analysis with propensity score matching was conducted. Thirty-six patients (11.0%) were diagnosed with concurrent endocarditis (SIE) by means of transoesophageal echocardiography. In our cohort, the average age was 65.82 ± 4.12 years and 64.9% of patients were male. The incidence of prior cardiac or renal disease was significantly higher in the SIE group (coronary heart disease SIE n = 13/36 vs. S n = 57/292, p &amp;lt; 0.05 and chronic heart failure n = 11/36 vs. S n = 41/292, p &amp;lt; 0.05, chronic renal failure SIE n = 14/36 vs. S n = 55/292, p &amp;lt; 0.05). Complex interdisciplinary coordination and diagnostics lead to a significant delay in surgical intervention (S = 4.5 ± 4.5 days vs. SIE = 8.9 ± 9.5 days, p &amp;lt; 0.05). Mortality did not show statistically significant differences: S (13.4%) and SIE (19.1%). Time to diagnosis and treatment is a key to efficient treatment and patient safety. In order to counteract delayed therapy, we developed a novel therapy algorithm based on the analysis of treatment processes of the SIE group. We propose a clear therapy pathway to avoid frequently observed pitfalls and delays in diagnosis to improve patient care and outcome

Trace element evidence for anatexis at oceanic magma chamber roofs and the role of partial melts for contamination of fresh MORB

International audienceAt oceanic spreading centers, interactions between magma and hydrothermal convecting systems trigger major physical, thermal, and chemical exchanges. The two-pyroxene hornfels recovered from the base of the sheeted dike sequence at Integrated Ocean Drilling Program (IODP) Site 1256 (equatorial Eastern Pacific) are interpreted as a conducting boundary layer between the underlying axial melt lens and the hydrothermally cooled sheeted dikes. They are cut by numerous small, felsic veins, which were recently interpreted as a product of hydrous partial melting of sheeted dikes. Here, we present trace element compositions of products (melts and residues) of hydrous partial melting experiments using basalts and hornfels from IODP Site 1256 as starting material. The experimental products generated between 910 °C and 970 °C match the natural lithologies from Site 1256 in terms of major and trace element compositions. The compositions of the anatectic melts correspond to the compositions of the felsic veins, while the residual minerals match the compositions of the two-pyroxene hornfels, evidencing that hydrous partial melting is an important magmatic process in the gabbro/dike transition of fast-spreading mid-oceanic ridges. Our results complement previous experimental studies on anatectic processes occurring at the roof of the magma chambers from fast-spreading mid-ocean ridges. Moreover, calculations of mixing and assimilation fractional crystallization using the experimental partial melts as contaminant/assimilant showed that anatectic melts can only be a minor contributor to the contamination process

N4BP1 restricts HIV-1 and its inactivation by MALT1 promotes viral reactivation

宿主がHIV-1感染を抑制する新たなメカニズムの解明 --N4BP1によるRNA分解とその調節がウイルス再活性化を調節する--. 京都大学プレスリリース. 2019-05-29.RNA-modulating factors not only regulate multiple steps of cellular RNA metabolism, but also emerge as key effectors of the immune response against invading viral pathogens including human immunodeficiency virus type-1 (HIV-1). However, the cellular RNA-binding proteins involved in the establishment and maintenance of latent HIV-1 reservoirs have not been extensively studied. Here, we screened a panel of 62 cellular RNA-binding proteins and identified NEDD4-binding protein 1 (N4BP1) as a potent interferon-inducible inhibitor of HIV-1 in primary T cells and macrophages. N4BP1 harbours a prototypical PilT N terminus-like RNase domain and inhibits HIV-1 replication by interacting with and degrading viral mRNA species. Following activation of CD4+ T cells, however, N4BP1 undergoes rapid cleavage at Arg 509 by the paracaspase named mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1). Mutational analyses and knockout studies revealed that MALT1-mediated inactivation of N4BP1 facilitates the reactivation of latent HIV-1 proviruses. Taken together, our findings demonstrate that the RNase N4BP1 is an efficient restriction factor of HIV-1 and suggest that inactivation of N4BP1 by induction of MALT1 activation might facilitate elimination of latent HIV-1 reservoirs