Evaluation of the Relationships between Intestinal Regional Lymph Nodes and Immune Responses in Viral Infections in Children

Viral infections increase the risk of developing allergies in childhood, and disruption of mucosal homeostasis is presumed to be involved. However, no study has reported a role for viral infections in such disruption. In this study, we clarified the mechanism of immunoglobulin A (IgA) overproduction in viral infections. Autopsies were performed on 33 pediatric cases, IgA and interferon (IFN)β levels were measured, and histopathological and immunohistochemical examinations were conducted. Furthermore, we cultured human cells and measured IFNβ and IgA levels to examine the effect of viral infections on IgA production. Blood IgA levels in viral infections were higher than in bacterial infections. Moreover, IFNβ levels in most viral cases were below the detection limit. Cell culture revealed increased IgA in gastrointestinal lymph nodes, especially in Peyer’s patches, due to enhanced IFNβ after viral stimulation. Conversely, respiratory regional lymph nodes showed enhanced IgA with no marked change in IFNβ. Overproduction of IgA, identified as an aberration of the immune system and resulting from excessive viral infection-induced IFNβ was observed in the intestinal regional lymph nodes, particularly in Peyer’s patches. Further, increased IgA without elevated IFNβ in the respiratory system suggested the possibility of a different mechanism from the gastrointestinal system.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

5' modifications improve potency and efficacy of DNA donors for precision genome editing

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

Mitochondrial Translation Occurs Preferentially in the Peri-Nuclear Mitochondrial Network of Cultured Human Cells

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in the distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

Mitochondrial translation occurs preferentially in the peri-nuclear mitochondrial network of cultured human cells.

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

A novel automated image analysis pipeline for quantifying morphological changes to the endoplasmic reticulum in cultured human cells

Background In mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm. This organelle is of particular interest to biologists, as its dysfunction is associated with numerous diseases, which often manifest themselves as changes to the structure and organisation of the reticular network. Due to its complex morphology, image analysis methods to quantitatively describe this organelle, and importantly any changes to it, are lacking. Results In this work we detail a methodological approach that utilises automated high-content screening microscopy to capture images of cells fluorescently-labelled for various ER markers, followed by their quantitative analysis. We propose that two key metrics, namely the area of dense ER and the area of polygonal regions in between the reticular elements, together provide a basis for measuring the quantities of rough and smooth ER, respectively. We demonstrate that a number of different pharmacological perturbations to the ER can be quantitatively measured and compared in our automated image analysis pipeline. Furthermore, we show that this method can be implemented in both commercial and open-access image analysis software with comparable results. Conclusions We propose that this method has the potential to be applied in the context of large-scale genetic and chemical perturbations to assess the organisation of the ER in adherent cell cultures.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

TCERG1L allelic variation is associated with cisplatin-induced hearing loss in childhood cancer, a PanCareLIFE study

In children with cancer, the heterogeneity in ototoxicity occurrence after similar treatment suggests a role for genetic susceptibility. Using a genome-wide association study (GWAS) approach, we identified a genetic variant in TCERG1L (rs893507) to be associated with hearing loss in 390 non-cranial irradiated, cisplatin-treated children with cancer. These results were replicated in two independent, similarly treated cohorts (n = 192 and 188, respectively) (combined cohort: P = 5.3 × 10−10, OR 3.11, 95% CI 2.2–4.5). Modulating TCERG1L expression in cultured human cells revealed significantly altered cellular responses to cisplatin-induced cytokine secretion and toxicity. These results contribute to insights into the genetic and pathophysiological basis of cisplatin-induced ototoxicity.

Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner

Stress granules are dynamic, reversible condensates composed of RNA and protein that assemble in eukaryotic cells in response to a variety of stressors and are normally disassembled after stress is removed. The composition and assembly of stress granules is well understood, but little is known about the mechanisms that govern disassembly. Impaired disassembly has been implicated in some diseases including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Using cultured human cells, we found that stress granule disassembly was context-dependent: Specifically in the setting of heat shock, disassembly required ubiquitination of G3BP1, the central protein within the stress granule RNA-protein network. We found that ubiquitinated G3BP1 interacted with the endoplasmic reticulum–associated protein FAF2, which engaged the ubiquitin-dependent segregase p97/VCP (valosin-containing protein). Thus, targeting of G3BP1 weakened the stress granule–specific interaction network, resulting in granule disassembly.