Global mapping of RNA homodimers in living cells

RNA homodimerization is important for various physiological processes, including the assembly of membraneless organelles, RNA subcellular localization, and packaging of viral genomes. However, understanding RNA dimerization has been hampered by the lack of systematic in vivo detection methods. Here, we show that CLASH, PARIS, and other RNA proximity ligation methods detect RNA homodimers transcriptome-wide as “overlapping” chimeric reads that contain more than one copy of the same sequence. Analyzing published proximity ligation data sets, we show that RNA:RNA homodimers mediated by direct base-pairing are rare across the human transcriptome, but highly enriched in specific transcripts, including U8 snoRNA, U2 snRNA, and a subset of tRNAs. Mutations in the homodimerization domain of U8 snoRNA impede dimerization in vitro and disrupt zebrafish development in vivo, suggesting an evolutionarily conserved role of this domain. Analysis of virus-infected cells reveals homodimerization of SARS-CoV-2 and Zika genomes, mediated by specific palindromic sequences located within protein-coding regions of N gene in SARS-CoV-2 and NS2A gene in Zika. We speculate that regions of viral genomes involved in homodimerization may constitute effective targets for antiviral therapies

Effects of zinc and fluoride on the remineralisation of artificial carious lesions under simulated plaque fluid conditions.

The aim was to study the effects of zinc (Zn) and fluoride (F) on remineralisation at plaque fluid concentrations. Artificial carious lesions were created in 2 acid-gel demineralising systems (initially infinitely undersaturated and partially saturated with respect to enamel) giving lesions with different mineral distribution characteristics (high and low R values, respectively) but similar integrated mineral loss values. Lesions of both types were assigned to 1 of 4 groups and remineralised for 5 days at 37°C. Zn and F were added, based on plaque fluid concentrations 1 h after application, to give 4 treatments: 231 μmol/l Zn, 10.5 μmol/l F, Zn/F combined and an unmodified control solution (non-F/non-Zn). Subsequently remineralisation was measured using microradiography. High-R lesions were analysed for calcium, phosphorus, F and Zn using electron probe micro-analysis. All lesions underwent statistically significant remineralisation. For low-R lesions, remineralisation was in the order F(a) < non-F/non-Zn(a) < Zn(a, b) < Zn/F(b), and for high-R lesions F(a) < non-F/non-Zn(b) < Zn(b) < Zn/F(c) (treatments with the same superscript letter not significantly different, at p < 0.05). Qualitatively, remineralisation occurred throughout non-F/non-Zn and Zn groups, predominantly at the surface zone (F) and within the lesion body (Zn/F). Electron probe micro-analysis revealed Zn in relatively large amounts in the outer regions (Zn, Zn/F). F was abundant not only at the surface (F), but also in the lesion body (Zn/F). Calcium:phosphate ratios were similar to hydroxyapatite (all). To conclude, under static remineralising conditions simulating plaque fluid, Zn/F treatment gave significantly greater remineralisation than did F treatment, possibly because Zn in the Zn/F group maintained greater surface zone porosity compared with F, facilitating greater lesion body remineralisation

Osteoblasts contribute to a protective niche that supports melanoma cell proliferation and survival

Melanoma is the deadliest form of skin cancer; a primary driver of this high level of morbidity is the propensity of melanoma cells to metastasize. When malignant tumours develop distant metastatic lesions the new local tissue niche is known to impact on the biology of the cancer cells. However, little is known about how different metastatic tissue sites impact on frontline targeted therapies. Intriguingly, melanoma bone lesions have significantly lower response to BRAF or MEK inhibitor therapies. Here, we have investigated how the cellular niche of the bone can support melanoma cells by stimulating growth and survival via paracrine signalling between osteoblasts and cancer cells. Melanoma cells can enhance the differentiation of osteoblasts leading to increased production of secreted ligands, including RANKL. Differentiated osteoblasts in turn can support melanoma cell proliferation and survival via the secretion of RANKL that elevates the levels of the transcription factor MITF, even in the presence of BRAF inhibitor. By blocking RANKL signalling, either via neutralizing antibodies, genetic alterations or the RANKL receptor inhibitor SPD304, the survival advantage provided by osteoblasts could be overcome

Using zebrafish larval models to study brain injury, locomotor and neuroinflammatory outcomes following intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a devastating condition with limited treatment options, and current understanding of pathophysiology is incomplete. Spontaneous cerebral bleeding is a characteristic of the human condition that has proven difficult to recapitulate in existing pre-clinical rodent models. Zebrafish larvae are frequently used as vertebrate disease models and are associated with several advantages, including high fecundity, optical translucency and non-protected status prior to 5 days post-fertilisation. Furthermore, other groups have shown that zebrafish larvae can exhibit spontaneous ICH. The aim of this study was to investigate whether such models can be utilised to study the pathological consequences of bleeding in the brain, in the context of pre-clinical ICH research. Here, we compared existing genetic (bubblehead) and chemically inducible (atorvastatin) zebrafish larval models of spontaneous ICH and studied the subsequent disease processes. Through live, non-invasive imaging of transgenic fluorescent reporter lines and behavioural assessment we quantified brain injury, locomotor function and neuroinflammation following ICH. We show that ICH in both zebrafish larval models is comparable in timing, frequency and location. ICH results in increased brain cell death and a persistent locomotor deficit. Additionally, in haemorrhaged larvae we observed a significant increase in macrophage recruitment to the site of injury. Live in vivo imaging allowed us to track active macrophage-based phagocytosis of dying brain cells 24 hours after haemorrhage. Morphological analyses and quantification indicated that an increase in overall macrophage activation occurs in the haemorrhaged brain. Our study shows that in zebrafish larvae, bleeding in the brain induces quantifiable phenotypic outcomes that mimic key features of human ICH. We hope that this methodology will enable the pre-clinical ICH community to adopt the zebrafish larval model as an alternative to rodents, supporting future high throughput drug screening and as a complementary approach to elucidating crucial mechanisms associated with ICH pathophysiology

Regulation of immune responses in primary biliary cholangitis: a transcriptomic analysis of peripheral immune cells

BACKGROUND AIMS: In patients with primary biliary cholangitis (PBC), the serum liver biochemistry measured during treatment with ursodeoxycholic acid-the UDCA response-accurately predicts long-term outcome. Molecular characterization of patients stratified by UDCA response can improve biological understanding of the high-risk disease, thereby helping to identify alternative approaches to disease-modifying therapy. In this study, we sought to characterize the immunobiology of the UDCA response using transcriptional profiling of peripheral blood mononuclear cell subsets. METHODS: We performed bulk RNA-sequencing of monocytes and TH1, TH17, TREG, and B cells isolated from the peripheral blood of 15 PBC patients with adequate UDCA response ("responders"), 16 PBC patients with inadequate UDCA response ("nonresponders"), and 15 matched controls. We used the Weighted Gene Co-expression Network Analysis to identify networks of co-expressed genes ("modules") associated with response status and the most highly connected genes ("hub genes") within them. Finally, we performed a Multi-Omics Factor Analysis of the Weighted Gene Co-expression Network Analysis modules to identify the principal axes of biological variation ("latent factors") across all peripheral blood mononuclear cell subsets. RESULTS: Using the Weighted Gene Co-expression Network Analysis, we identified modules associated with response and/or disease status (q<0.05) in each peripheral blood mononuclear cell subset. Hub genes and functional annotations suggested that monocytes are proinflammatory in nonresponders, but antiinflammatory in responders; TH1 and TH17 cells are activated in all PBC cases but better regulated in responders; and TREG cells are activated-but also kept in check-in responders. Using the Multi-Omics Factor Analysis, we found that antiinflammatory activity in monocytes, regulation of TH1 cells, and activation of TREG cells are interrelated and more prominent in responders. CONCLUSIONS: We provide evidence that adaptive immune responses are better regulated in patients with PBC with adequate UDCA response

RAS at the Golgi antagonizes malignant transformation through PTPRκ-mediated inhibition of ERK activation

© The Author(s) 2018.RAS GTPases are frequently mutated in human cancer. H- and NRAS isoforms are distributed over both plasma-membrane and endomembranes, including the Golgi complex, but how this organizational context contributes to cellular transformation is unknown. Here we show that RAS at the Golgi is selectively activated by apoptogenic stimuli and antagonizes cell survival by suppressing ERK activity through the induction of PTPRκ, which targets CRAF for dephosphorylation. Consistently, in contrast to what occurs at the plasma-membrane, RAS at the Golgi cannot induce melanoma in zebrafish. Inactivation of PTPRκ, which occurs frequently in human melanoma, often coincident with TP53 inactivation, accelerates RAS-ERK pathway-driven melanomagenesis in zebrafish. Likewise, tp53 disruption in zebrafish facilitates oncogenesis driven by RAS from the Golgi complex. Thus, RAS oncogenic potential is strictly dependent on its sublocalization, with Golgi complex-located RAS antagonizing tumor development.We are grateful to Drs: Ignacio Rubio, Yardena Samuels, Mariano Barbacid and Javier León for providing reagents; and Alicia Noriega, Sandra Zunzunegui y Victor Campa for technical support. Crespo laboratory is supported by grant SAF-2015 63638R (MINECO/ FEDER, UE); by Red Temática de Investigación Cooperativa sobre el Cáncer (RTICC). RD/12/0036/0033 and by Asociación Española Contra el Cáncer (AECC), grant GCB141423113. Work in the Hurlstone laboratory was unded by a grant from the European Research Council (ERC-2011-StG-282059 PROMINENT). B.C. is supported by a Retos Jóvenes Investigadores grant SAF2015-73364-JIN (AEI/FEDER, UE) and a grant from Fundación Francisco Cobos. X.R.B. is supported by grants from the CastillaLeón Government (BIO/SA01/15, CSI049U16), MINECO (SAF2015-64556-R, RD12/ 0036/0002), Worldwide Cancer Research (14-1248), Ramón Areces Foundation, andAECC (GC16173472GARC). Spanish funding to P.C., B.C., and X.R.B. is partially supported by the European Regional Development Fund

Bacterial and metabolic phenotypes associated with inadequate response to ursodeoxycholic acid treatment in primary biliary cholangitis

Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease with ursodeoxycholic acid (UDCA) as first-line treatment. Poor response to UDCA is associated with a higher risk of progressing to cirrhosis, but the underlying mechanisms are unclear. UDCA modulates the composition of primary and bacterial-derived bile acids (BAs). We characterized the phenotypic response to UDCA based on BA and bacterial profiles of PBC patients treated with UDCA. Patients from the UK-PBC cohort (n = 419) treated with UDCA for a minimum of 12-months were assessed using the Barcelona dynamic response criteria. BAs from serum, urine, and feces were analyzed using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry and fecal bacterial composition measured using 16S rRNA gene sequencing. We identified 191 non-responders, 212 responders, and a subgroup of responders with persistently elevated liver biomarkers (n = 16). Responders had higher fecal secondary and tertiary BAs than non-responders and lower urinary bile acid abundances, with the exception of 12-dehydrocholic acid, which was higher in responders. The sub-group of responders with poor liver function showed lower alpha-diversity evenness, lower abundance of fecal secondary and tertiary BAs than the other groups and lower levels of phyla with BA-deconjugation capacity (Actinobacteriota/Actinomycetota, Desulfobacterota, Verrucomicrobiota) compared to responders. UDCA dynamic response was associated with an increased capacity to generate oxo-/epimerized secondary BAs. 12-dehydrocholic acid is a potential biomarker of treatment response. Lower alpha-diversity and lower abundance of bacteria with BA deconjugation capacity might be associated with an incomplete response to treatment in some patients

Regulation of immune responses in primary biliary cholangitis: a transcriptomic analysis of peripheral immune cells

Copyright \ua9 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.Background &amp; Aims: In patients with primary biliary cholangitis (PBC), the serum liver biochemistry measured during treatment with ursodeoxycholic acid-the UDCA response-accurately predicts long-term outcome. Molecular characterization of patients stratified by UDCA response can improve biological understanding of the high-risk disease, thereby helping to identify alternative approaches to disease-modifying therapy. In this study, we sought to characterize the immunobiology of the UDCA response using transcriptional profiling of peripheral blood mononuclear cell subsets. Methods: We performed bulk RNA-sequencing of monocytes and TH1, TH17, TREG, and B cells isolated from the peripheral blood of 15 PBC patients with adequate UDCA response (“responders”), 16 PBC patients with inadequate UDCA response (“nonresponders”), and 15 matched controls. We used the Weighted Gene Co-expression Network Analysis to identify networks of co-expressed genes (“modules”) associated with response status and the most highly connected genes (“hub genes”) within them. Finally, we performed a Multi-Omics Factor Analysis of the Weighted Gene Co-expression Network Analysis modules to identify the principal axes of biological variation (“latent factors”) across all peripheral blood mononuclear cell subsets. Results: Using the Weighted Gene Co-expression Network Analysis, we identified modules associated with response and/or disease status (q &lt; 0.05) in each peripheral blood mononuclear cell subset. Hub genes and functional annotations suggested that monocytes are proinflammatory in nonresponders, but antiinflammatory in responders; TH1 and TH17 cells are activated in all PBC cases but better regulated in responders; and TREG cells are activated-but also kept in check-in responders. Using the Multi-Omics Factor Analysis, we found that antiinflammatory activity in monocytes, regulation of TH1 cells, and activation of TREG cells are interrelated and more prominent in responders. Conclusions: We provide evidence that adaptive immune responses are better regulated in patients with PBC with adequate UDCA response

Regulation of immune responses in primary biliary cholangitis: a transcriptomic analysis of peripheral immune cells

BACKGROUND AIMS: In patients with primary biliary cholangitis (PBC), the serum liver biochemistry measured during treatment with ursodeoxycholic acid-the UDCA response-accurately predicts long-term outcome. Molecular characterization of patients stratified by UDCA response can improve biological understanding of the high-risk disease, thereby helping to identify alternative approaches to disease-modifying therapy. In this study, we sought to characterize the immunobiology of the UDCA response using transcriptional profiling of peripheral blood mononuclear cell subsets. METHODS: We performed bulk RNA-sequencing of monocytes and TH1, TH17, TREG, and B cells isolated from the peripheral blood of 15 PBC patients with adequate UDCA response ("responders"), 16 PBC patients with inadequate UDCA response ("nonresponders"), and 15 matched controls. We used the Weighted Gene Co-expression Network Analysis to identify networks of co-expressed genes ("modules") associated with response status and the most highly connected genes ("hub genes") within them. Finally, we performed a Multi-Omics Factor Analysis of the Weighted Gene Co-expression Network Analysis modules to identify the principal axes of biological variation ("latent factors") across all peripheral blood mononuclear cell subsets. RESULTS: Using the Weighted Gene Co-expression Network Analysis, we identified modules associated with response and/or disease status (q<0.05) in each peripheral blood mononuclear cell subset. Hub genes and functional annotations suggested that monocytes are proinflammatory in nonresponders, but antiinflammatory in responders; TH1 and TH17 cells are activated in all PBC cases but better regulated in responders; and TREG cells are activated-but also kept in check-in responders. Using the Multi-Omics Factor Analysis, we found that antiinflammatory activity in monocytes, regulation of TH1 cells, and activation of TREG cells are interrelated and more prominent in responders. CONCLUSIONS: We provide evidence that adaptive immune responses are better regulated in patients with PBC with adequate UDCA response