Significant Variations in Double-Stranded RNA Levels in Cultured Skin Cells

\ua9 2024 by the authors.Endogenous double-stranded RNA has emerged as a potent stimulator of innate immunity. Under physiological conditions, endogenous dsRNA is maintained in the cell nucleus or the mitochondria; however, if protective mechanisms are breached, it leaches into the cytoplasm and triggers immune signaling pathways. Ectopic activation of innate immune pathways is associated with various diseases and senescence and can trigger apoptosis. Hereby, the level of cytoplasmic dsRNA is crucial. We have enriched dsRNA from two melanoma cell lines and primary dermal fibroblasts, including a competing probe, and analyzed the dsRNA transcriptome using RNA sequencing. There was a striking difference in read counts between the cell lines and the primary cells, and the effect was confirmed by northern blotting and immunocytochemistry. Both mitochondria (10–20%) and nuclear transcription (80–90%) contributed significantly to the dsRNA transcriptome. The mitochondrial contribution was lower in the cancer cells compared to fibroblasts. The expression of different transposable element families was comparable, suggesting a general up-regulation of transposable element expression rather than stimulation of a specific sub-family. Sequencing of the input control revealed minor differences in dsRNA processing pathways with an upregulation of oligoadenylate synthase and RNP125 that negatively regulates the dsRNA sensors RIG1 and MDA5. Moreover, RT-qPCR, Western blotting, and immunocytochemistry confirmed the relatively minor adaptations to the hugely different dsRNA levels. As a consequence, these transformed cell lines are potentially less tolerant to interventions that increase the formation of endogenous dsRNA

Microlaser-based contractility sensing in single cardiomyocytes and whole hearts

Microscopic whispering gallery mode lasers detect minute changes in cellular refractive index inside individual cardiac cells and in live zebrafish. We show that these signals encode cardiac contractility that can be used for intravital sensing.Postprin

A Suborbital Payload for Soft X-ray Spectroscopy of Extended Sources

We present a suborbital rocket payload capable of performing soft X-ray spectroscopy on extended sources. The payload can reach resolutions of ~100(lambda/dlambda) over sources as large as 3.25 degrees in diameter in the 17-107 angstrom bandpass. This permits analysis of the overall energy balance of nearby supernova remnants and the detailed nature of the diffuse soft X-ray background. The main components of the instrument are: wire grid collimators, off-plane grating arrays and gaseous electron multiplier detectors. This payload is adaptable to longer duration orbital rockets given its comparatively simple pointing and telemetry requirements and an abundance of potential science targets.Comment: Accepted to Experimental Astronomy, 12 pages plus 1 table and 17 figure

An aesthetic of the unknown

This paper begins to establish an 'aesthetic of the unknown' by drawing together theorists and approaches from mainstream art criticism to provide a starting-point for an aesthetic sympathetic with Jungian perspectives, in an attempt to bridge a gap between contemporary abstract painting, contemporary art theory and Jungian studies. This is a framework for approaching the abstract painting not as an object awaiting interpretation or 'reading', but rather as something that offers a numinous experience (or experience of the unknown), which can be thought about but may remain ultimately unknowable and irreducible. Such experience - involving both the unconscious and conscious mind - would provide glimpsesof forms of meaning not accessible to full rational exposition. This type of uncosciously understood meaning is explored, acknowledging that there is a need to preserve this encounter with the unknown and a need for a contemporary critical, theoretical framework that recognises the importance of this within abstract painting

Widespread formation of double-stranded RNAs in testis

The testis transcriptome is highly complex and includes RNAs that potentially hybridize to form double-stranded RNA (dsRNA). We isolated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testes of juvenile Dicer1 knockout mice, age-matched controls, and adult animals. Comparison of our data set with recently published data from mouse liver revealed that the dsRNA transcriptome in testis is markedly different from liver: In testis, dsRNA-forming transcripts derive from mRNAs including promoters and immediate downstream regions, whereas in somatic cells they originate more often from introns and intergenic transcription. The genes that generate dsRNA are significantly expressed in isolated male germ cells with particular enrichment in pachytene spermatocytes. dsRNA formation is lower on the sex (X and Y) chromosomes. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knockout of Dicer1 has only a minor effect on the total number of transcript peaks associated with dsRNA. The comparison between dsRNA-associated genes in testis and liver with a reported set of genes that produce endogenous siRNAs reveals a significant overlap in testis but not in liver. Testis dsRNAs also significantly associate with natural antisense genes-again, this feature is not observed in liver. These findings point to a testis-specific mechanism involving natural antisense transcripts and the formation of dsRNAs that feed into the RNA interference pathway, possibly to mitigate the mutagenic impacts of recombination and transposon mobilization.</p

TOP2B Is Required to Maintain the Adrenergic Neural Phenotype and for ATRA-Induced Differentiation of SH-SY5Y Neuroblastoma Cells.

The neuroblastoma cell line SH-SY5Y is widely used to study retinoic acid (RA)-induced gene expression and differentiation and as a tool to study neurodegenerative disorders. SH-SY5Y cells predominantly exhibit adrenergic neuronal properties, but they can also exist in an epigenetically interconvertible alternative state with more mesenchymal characteristics; as a result, these cells can be used to study gene regulation circuitry controlling neuroblastoma phenotype. Using a combination of pharmacological inhibition and targeted gene inactivation, we have probed the requirement for DNA topoisomerase IIB (TOP2B) in RA-induced gene expression and differentiation and in the balance between adrenergic neuronal versus mesenchymal transcription programmes. We found that expression of many, but not all genes that are rapidly induced by ATRA in SH-SY5Y cells was significantly reduced in the TOP2B null cells; these genes include BCL2, CYP26A1, CRABP2, and NTRK2. Comparing gene expression profiles in wild-type versus TOP2B null cells, we found that long genes and genes expressed at a high level in WT SH-SY5Y cells were disproportionately dependent on TOP2B. Notably, TOP2B null SH-SY5Y cells upregulated mesenchymal markers vimentin (VIM) and fibronectin (FN1) and components of the NOTCH signalling pathway. Enrichment analysis and comparison with the transcription profiles of other neuroblastoma-derived cell lines supported the conclusion that TOP2B is required to fully maintain the adrenergic neural-like transcriptional signature of SH-SY5Y cells and to suppress the alternative mesenchymal epithelial-like epigenetic state

Occupation of racial grief, loss as a resource : learning from ‘The Combahee River Collective Black Feminist Statement'

The methodology of ‘occupation’ through rereading The Combahee River Collective Black Feminist Statement (The Combahee River Collective, in: James, Sharpley-Whiting (eds) The Black Feminist Reader. Blackwell Publishers Ltd., Oxford, pp 261–270, 1977) demonstrates the necessity of temporal linkages to historical Black feminist texts and the wisdom of Black feminist situated knowers. This paper argues that racism produces grief and loss and as long as there is racism, we all remain in racial grief and loss. However, in stark contrast to the configuration of racial grief and loss as something to get over, perhaps grief and loss can be thought about differently, for example, in terms of racial grief and loss as a resource. This paper questions Western Eurocentric paternalistic responses to Black women’s ‘talk about their feelings of craziness… [under] patriarchal rule’ (The Combahee River Collective 1977: 262) and suggests alternative ways of thinking about the psychological impact of grief and loss in the context of racism. In this paper, a Black feminist occupation of racial grief and loss includes the act of residing within, and the act of working with the constituent elements of racial grief and loss. The proposal is that an occupation of racial grief and loss is a paradoxical catalyst for building a twenty-first century global intersectional Black feminist movement

Mild Transient Hypercapnia as a Novel Fear Conditioning Stimulus Allowing Re-Exposure during Sleep

Introduction:Studies suggest that sleep plays a role in traumatic memories and that treatment of sleep disorders may help alleviate symptoms of posttraumatic stress disorder. Fear-conditioning paradigms in rodents are used to investigate causal mechanisms of fear acquisition and the relationship between sleep and posttraumatic behaviors. We developed a novel conditioning stimulus (CS) that evoked fear and was subsequently used to study re-exposure to the CS during sleep.Methods:Experiment 1 assessed physiological responses to a conditioned stimulus (mild transient hypercapnia, mtHC; 3.0% CO2; n = 17)+footshock for the purpose of establishing a novel CS in male FVB/J mice. Responses to the novel CS were compared to tone+footshock (n = 18) and control groups of tone alone (n = 17) and mild transient hypercapnia alone (n = 10). A second proof of principle experiment re-exposed animals during sleep to mild transient hypercapnia or air (control) to study sleep processes related to the CS.Results:Footshock elicited a response of acute tachycardia (30-40 bpm) and increased plasma epinephrine. When tone predicted footshock it elicited mild hypertension (1-2 mmHg) and a three-fold increase in plasma epinephrine. When mtHC predicted footshock it also induced mild hypertension, but additionally elicited a conditioned bradycardia and a smaller increase in plasma epinephrine. The overall mean 24 hour sleep-wake profile was unaffected immediately after fear conditioning.Discussion:Our study demonstrates the efficacy of mtHC as a conditioning stimulus that is perceptible but innocuous (relative to tone) and applicable during sleep. This novel model will allow future studies to explore sleep-dependent mechanisms underlying maladaptive fear responses, as well as elucidate the moderators of the relationship between fear responses and sleep. © 2013 McDowell et al

Symptom-based stratification of patients with primary Sjögren's syndrome: multi-dimensional characterisation of international observational cohorts and reanalyses of randomised clinical trials

Background: Heterogeneity is a major obstacle to developing effective treatments for patients with primary Sjögren's syndrome. We aimed to develop a robust method for stratification, exploiting heterogeneity in patient-reported symptoms, and to relate these differences to pathobiology and therapeutic response. / Methods: We did hierarchical cluster analysis using five common symptoms associated with primary Sjögren's syndrome (pain, fatigue, dryness, anxiety, and depression), followed by multinomial logistic regression to identify subgroups in the UK Primary Sjögren's Syndrome Registry (UKPSSR). We assessed clinical and biological differences between these subgroups, including transcriptional differences in peripheral blood. Patients from two independent validation cohorts in Norway and France were used to confirm patient stratification. Data from two phase 3 clinical trials were similarly stratified to assess the differences between subgroups in treatment response to hydroxychloroquine and rituximab. / Findings: In the UKPSSR cohort (n=608), we identified four subgroups: Low symptom burden (LSB), high symptom burden (HSB), dryness dominant with fatigue (DDF), and pain dominant with fatigue (PDF). Significant differences in peripheral blood lymphocyte counts, anti-SSA and anti-SSB antibody positivity, as well as serum IgG, κ-free light chain, β2-microglobulin, and CXCL13 concentrations were observed between these subgroups, along with differentially expressed transcriptomic modules in peripheral blood. Similar findings were observed in the independent validation cohorts (n=396). Reanalysis of trial data stratifying patients into these subgroups suggested a treatment effect with hydroxychloroquine in the HSB subgroup and with rituximab in the DDF subgroup compared with placebo. / Interpretation: Stratification on the basis of patient-reported symptoms of patients with primary Sjögren's syndrome revealed distinct pathobiological endotypes with distinct responses to immunomodulatory treatments. Our data have important implications for clinical management, trial design, and therapeutic development. Similar stratification approaches might be useful for patients with other chronic immune-mediated diseases. / Funding: UK Medical Research Council, British Sjogren's Syndrome Association, French Ministry of Health, Arthritis Research UK, Foundation for Research in Rheumatology