Viruses and the cellular RNA decay machinery.

The ability to control cellular and viral gene expression, either globally or selectively, is central to a successful viral infection, and it is also crucial for the host to respond and eradicate pathogens. In eukaryotes, regulation of message stability contributes significantly to the control of gene expression and plays a prominent role in the normal physiology of a cell as well as in its response to environmental and pathogenic stresses. Not surprisingly, emerging evidence indicates that there are significant interactions between the eukaryotic RNA turnover machinery and a wide variety of viruses. Interestingly, in many cases viruses have evolved mechanisms not only to evade eradication by these pathways, but also to manipulate them for enhanced viral replication and gene expression. Given our incomplete understanding of how many of these pathways are normally regulated, viruses should be powerful tools to help deconstruct the complex networks and events governing eukaryotic RNA stability

Constant rate of p53 tetramerization in response to DNA damage controls the p53 response

The dynamics of the tumor suppressor protein p53 have been previously investigated in single cells using fluorescently tagged p53. Such approach reports on the total abundance of p53 but does not provide a measure for functional p53. We used fluorescent protein-fragment complementation assay (PCA) to quantify in single cells the dynamics of p53 tetramers, the functional units of p53. We found that while total p53 increases proportionally to the input strength, p53 tetramers are formed in cells at a constant rate. This breaks the linear input–output relation and dampens the p53 response. Disruption of the p53-binding protein ARC led to a dose-dependent rate of tetramers formation, resulting in enhanced tetramerization and induction of p53 target genes. Our work suggests that constraining the p53 response in face of variable inputs may protect cells from committing to terminal outcomes and highlights the importance of quantifying the active form of signaling molecules in single cells. Quantification of the dynamics of p53 tetramers in single cells using a fluorescent protein-fragment complementation assay reveals that, while total p53 increases proportionally to the DNA damage strength, p53 tetramers are formed at a constant rate

Dynamics of p53 tetramers in live single cells

Protein homo-oligomerization is the process through which identical peptides bind together to form higher order complexes. Self-interactions in many cases are constitutive and stable, used as building blocks for biological structures, such as rings, filaments and membranes. Further, homo-oligomerization can also be a regulatory process that influences the proteins' function such as change in transcriptional activities for transcription factors. Innovative methods to measure oligomerization in live cells are needed in order to understand regulation and function of homooligomerization in the native cellular context. This thesis examines the case of the tumor suppressor p53, whose homo-tetramerization greatly influences its activity as a transcription factor. We develop methods to quantify p53's self-interaction in individual living cells and follow it in time after DNA damage. The two methods we developed have complementary qualities and different applications. We first use fluorescent correlation spectroscopy to study the molecular events occurring in the first three hours of the p53 in response to double strand breaks. We find that in the absence of stress p53 is present in a mixture of, monomers, dimers and tetramers. When damage is sensed, oligomerization is rapidly induced and nearly all p53 is found bound in tetramers. We combine our data with a mathematical framework to propose the existence of a dedicated mechanism triggering p53 oligomerization independently of protein stabilization. Next, we use bimolecular fluorescent complementation to probe for tetramerization in the longer timescales of p53's response to ultraviolet radiation. In this context we find that even though the rate of p53 accumulation increases with the dose of radiation, p53 tetramers are formed at a steady rate. We hence propose the existence of an inhibitory mechanism that prevents the oligomerization reaction from following a linear input-output relation. We identify ARC, a known cofactor of p53, as part of this inhibitory mechanism. Downregulation of ARC restore the linear relation between to total and tetrameric p53. Finally, in both experimental setups higher oligomerization lead to an increase in p53 activity, underscoring the connection between regulation of oligomerization and the transcriptional activity of p53 in cancer cells. Collectively, this work emphasizes the importance of precise measurements to investigate the regulation and function of higher order complexes and provides generally applicable methods to quantify homo-oligomerization in live single cells

Brain-derived neurotrophic factor (BDNF), B-catenin, and cortisol levels correlated with the severity of adverse childhood experiences (ACEs) score in patients with Schizophrenia spectrum disorders

Psychosis, as seen in Schizophrenia and Schizoaffective disorder, is the most debilitating of psychiatric dysfunctions, affecting more than 21 million people per year globally.Adverse childhood experiences (ACE) and psychosis have significant correlation. We used the ACE Questionnaire (ACEQ) in this study to evaluate childhood trauma. There are, however, many instruments used to assess ACEs. Instruments vary and are used at the discretion of the study facilitator in order to meet the perceived needs of the study. We assessed the prevalence of the instruments used to assess childhood trauma in individuals with psychosis via a systematic review.BDNF and B-catenin are critical in early and continued neuronal development and proliferation, particularly in the hippocampus (Hpc). Cortisol, well-known for being the major stress hormone, and is known to damage to Hpc neurons in chronic stress. In this study serum BDNF, B-catenin and cortisol will be measured in patients with a diagnosis of Schizophrenia (Sz) or Schizoaffective disorder (SzA) and evaluated for correlation with their respective ACEs scores.We had a sample size of 11 patients (4 Female, 7 Male) with diagnosis of either Sz or SzA (Sz=2M, SzA=4F, 5M). All were administered a Structured Clinical Interview for DSM-5 - Clinician's Version (SCID-5-CV) to confirm diagnoses, the ACEQ, and a blood draw to evaluate serum for biomarkers.We found a significant negative correlation (p=0.027) between cortisol levels and higher ACEQ scores in our sample population. B-catenin and BDNF demonstrated downward linear trends relative to higher ACEQ scores, which with increased power could show significant correlation.170 studies met criteria for the systematic review, 24 instruments were isolated from those studies. Of those 100, (58.9%), of the studies isolated utilized the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences in patients with psychosis

Two p53 tetramers bind one consensus DNA response element

p53 tumor suppressor is a transcription factor that controls cell cycle and genetic integrity. In response to genotoxic stress p53 activates DNA repair, cell cycle arrest, apoptosis or senescence, which are initiated via p53 binding to its specific DNA response elements (RE). The consensus p53 DNA RE consists of two decameric palindromic half-site sequences. Crystallographic studies have demonstrated that two isolated p53 DNA-binding core domains interact with one half-site of the p53 DNA REs suggesting that one p53 tetramer is bound to one RE. However, our recent 3D cryo-EM studies showed that the full-length p53 tetramer is bound to only one half-site of RE. Here, we have used biochemical and electron microscopy (EM) methods to analyze DNA-binding of human and murine p53 tetramers to various p53 DNA REs. Our new results demonstrate that two p53 tetramers can interact sequence-specifically with one DNA RE at the same time. In particular, the EM structural analysis revealed that two p53 tetramers bind one DNA RE simultaneously with DNA positioned between them. These results demonstrate a mode different from that assumed previously for the p53-DNA interaction and suggest important biological implications on p53 activity as a transcriptional regulator of cellular response to stress

Caspase-Mediated Regulation and Cellular Heterogeneity of the cGAS/STING Pathway in Kaposi’s Sarcoma-Associated Herpesvirus Infection.

As a result of the ongoing virus-host arms race, viruses have evolved numerous immune subversion strategies, many of which are aimed at suppressing the production of type I interferons (IFNs). Apoptotic caspases have recently emerged as important regulators of type I IFN signaling both in noninfectious contexts and during viral infection. Despite being widely considered antiviral factors since they can trigger cell death, several apoptotic caspases promote viral replication by suppressing innate immune response. Indeed, we previously discovered that the AIDS-associated oncogenic gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) exploits caspase activity to suppress the antiviral type I IFN response and promote viral replication. However, the mechanism of this novel viral immune evasion strategy is poorly understood, particularly with regard to how caspases antagonize IFN signaling during KSHV infection. Here, we show that caspase activity inhibits the DNA sensor cGAS during KSHV lytic replication to block type I IFN induction. Furthermore, we used single-cell RNA sequencing to reveal that the potent antiviral state conferred by caspase inhibition is mediated by an exceptionally small percentage of IFN-β-producing cells, thus uncovering further complexity of IFN regulation during viral infection. Collectively, these results provide insight into multiple levels of cellular type I IFN regulation that viruses co-opt for immune evasion. Unraveling these mechanisms can inform targeted therapeutic strategies for viral infections and reveal cellular mechanisms of regulating interferon signaling in the context of cancer and chronic inflammatory diseases.post-print1848 K

Skills use and common treatment processes in dialectical behaviour therapy for borderline personality disorder

Background and Objectives Dialectical behaviour therapy (DBT) trains participants to use behavioural skills for managing their emotions. The study aimed to evaluate whether skills use is associated with positive treatment outcomes independently of treatment processes that are common across different therapeutic models. Method Use of the DBT skills and three common treatment processes (therapeutic alliance, treatment credibility and self-efficacy) were assessed every 2 months for a year in 70 individuals with borderline personality disorder receiving DBT. Mixed-multilevel modelling was used to determine the association of these factors with frequency of self-harm and with treatment dropout. Results Participants who used the skills less often at any timepoint were more likely to drop out of DBT in the subsequent two months, independently of their self-efficacy, therapeutic alliance or perceived treatment credibility. More frequent use of the DBT skills and higher self-efficacy were each independently associated with less frequent concurrent self-harm. Treatment credibility and the alliance were not independently associated with self-harm or treatment dropout. Limitations The skills use measure could not be applied to a control group who did not receive DBT. The sample size was insufficient for structural equation modelling. Conclusion Practising the DBT skills and building an increased sense of self-efficacy may be important and partially independent treatment processes in dialectical behaviour therapy. However, the direction of the association between these variables and self-harm requires further evaluation