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The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases., (© 2024. The Author(s).)

The clinical importance and the pathogenesis of the MW and STL polyomaviruses (PyVs) remain unclear. Our aim was to study the seroprevalence of MWPyV and STLPyV, and to examine the prevalence of viral DNA in respiratory samples and secondary lymphoid tissues. In total, 618 serum samples (0.8-90 years) were analyzed for seroprevalence. For the DNA prevalence study, 146 patients (2.5-37.5 years) were sampled for adenoids (n = 100), tonsils (n = 100), throat swabs (n = 146), and middle ear discharge (n = 15) in study Group 1. In Group 2, we analyzed 1130 nasopharyngeal samples from patients (0.8-92 years) tested for SARS-CoV-2 infection. The adult seropositivity was 54% for MWPyV, and 81.2% for STLPyV. Both seroprevalence rates increased with age; however, the majority of STLPyV primary infections appeared to occur in children. MWPyV was detected in 2.7%-4.9% of respiratory samples, and in a middle ear discharge. STLPyV DNA prevalence was 1.4%-3.4% in swab samples, and it was detected in an adenoid and in a middle ear discharge. The prevalence of both viruses was significantly higher in the children. Noncoding control regions of both viruses and the complete genomes of STLPyV were sequenced. MWPyV and STLPyV are widespread viruses, and respiratory transmission may be possible., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Antigen-specific B-cell ELISPOT and multicolor FluoroSpot assays, in which the membrane-bound antigen itself serves as the capture reagent for the antibodies that B cells secrete, inherently result in a broad range of spot sizes and intensities. The diversity of secretory footprint morphologies reflects the polyclonal nature of the antigen-specific B cell repertoire, with individual antibody-secreting B cells in the test sample differing in their affinity for the antigen, fine epitope specificity, and activation/secretion kinetics. To account for these heterogeneous spot morphologies, and to eliminate the need for setting up subjective counting parameters well-by-well, CTL introduces here its cutting-edge deep learning-based IntelliCount ™ algorithm within the ImmunoSpot ® Studio Software Suite, which integrates CTL's proprietary deep neural network. Here, we report detailed analyses of spots with a broad range of morphologies that were challenging to analyze using standard parameter-based counting approaches. IntelliCount ™ , especially in conjunction with high dynamic range (HDR) imaging, permits the extraction of accurate, high-content information of such spots, as required for assessing the affinity distribution of an antigen-specific memory B-cell repertoire ex vivo. IntelliCount ™ also extends the range in which the number of antibody-secreting B cells plated and spots detected follow a linear function; that is, in which the frequencies of antigen-specific B cells can be accurately established. Introducing high-content analysis of secretory footprints in B-cell ELISPOT/FluoroSpot assays, therefore, fundamentally enhances the depth in which an antigen-specific B-cell repertoire can be studied using freshly isolated or cryopreserved primary cell material, such as peripheral blood mononuclear cells., (© 2024. The Author(s).)