Spt5 Cooperates with Human Immunodeficiency Virus Type 1 Tat by Preventing Premature RNA Release at Terminator Sequences

The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), a protein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important role in late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongation factor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5

The role of membrane-bound metal ions in toxicity of a human cancer cell-active pore-forming toxin Cry41Aa from Bacillus thuringiensis

Bacillus thuringiensis crystal (Cry) proteins, used for decades as insecticidal toxins, are well known to be toxic to certain insects, but not to mammals. A novel group of Cry toxins called parasporins possess a strong cytocidal activity against some human cancer cells. Cry41Aa, or parasporin3, closely resembles commercially used insecticidal toxins and yet is toxic to the human hepatic cancer cell line HepG2, disrupting membranes of susceptible cells, similar to its insecticidal counterparts. In this study, we explore the protective effect that the common divalent metal chelator EGTA exerts on Cry41Aa's activity on HepG2 cells. Our results indicate that rather than interfering with a signalling pathway as a result of chelating cations in the medium, the chelator prevented the toxin's interaction with the membrane, and thus the subsequent steps of membrane damage and p38 phosphorylation, by removing cations bound to plasma membrane components. BAPTA and DTPA also inhibited Cry41Aa toxicity but at higher concentrations. We also show for the first time that Cry41Aa induces pore formation in planar lipid bilayers. This activity is not altered by EGTA, consistent with a biological context of chelation. Salt supplementation assays identified Ca2+, Mn2+ and Zn2+ as being able to reinstate Cry41Aa activity. Our data suggest the existence of one or more metal cation-dependent receptors in the Cry41Aa mechanism of action

The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts

Understanding how certain protein toxins from the normally insecticidal bacterium Bacillus thuringiensis (Bt) target human cell lines has implications for both the risk assessment of products containing these toxins and potentially for cancer therapy. This understanding requires knowledge of whether the human cell active toxins work by the same mechanism as their insecticidal counterparts or by alternative ones. The Bt Cry41Aa (also known as Parasporin3) toxin is structurally related to the toxins synthesised by commercially produced transgenic insect-resistant plants, with the notable exception of an additional C-terminal β-trefoil ricin domain. To better understand its mechanism of action, we developed an efficient expression system for the toxin and created mutations in regions potentially involved in the toxic mechanism. Deletion of the ricin domain did not significantly affect the activity of the toxin against the human HepG2 cell line, suggesting that this region was not responsible for the mammalian specificity of Cry41Aa. Various biochemical assays suggested that unlike some other human cell active toxins from Bt Cry41Aa did not induce apoptosis, but that its mechanism of action was consistent with that of a pore-forming toxin. The toxin induced a rapid and significant decrease in metabolic activity. Adenosine triphosphate depletion, cell swelling and membrane damage were also observed. An exposed loop region believed to be involved in receptor binding of insecticidal Cry toxins was shown to be important for the activity of Cry41Aa against HepG2 cells

Enhancer control of MicroRNA miR-155 expression in EpsteinBarr virus-infected B cells

The oncogenic microRNA miR-155 is the most frequently upregulated miRNA in Epstein-Barr virus (EBV)-positive B cell malignancies and is upregulated in other non-viral lymphomas. Both the EBV nuclear antigen 2 (EBNA2), and B cell transcription factor, interferon regulatory factor 4 (IRF4) are known to activate transcription of the host cell gene from which miR-155 is processed (miR-155HG, BIC). EBNA2 also activates IRF4 transcription indicating that EBV may upregulate miR-155 through direct and indirect mechanisms. The mechanism of transcriptional regulation of IRF4 and miR-155HG by EBNA2 however has not been defined. We demonstrate that EBNA2 can activate IRF4 and miR-155HG expression through specific upstream enhancers that are dependent on the Notch signaling transcription factor RBPJ, a known binding partner of EBNA2. We demonstrate that in addition to activation of the miR-155HG promoter, IRF4 can also activate miR-155HG via the upstream enhancer also targeted by EBNA2. Gene editing to remove the EBNA2- and IRF4-responsive miR-155HG enhancer located 60 kb upstream of miR-155HG led to reduced miR155HG expression in EBV-infected cells. Our data therefore demonstrate that specific RBPJ-dependent enhancers regulate the IRF4-miR-155 expression network and play a key role in the maintenance of miR-155 expression in EBV-infected B cells. These findings provide important insights that will improve our understanding of miR-155 control in B cell malignancies

Declaring a Patient Brain Dead on Extracorporeal Membrane Oxygenation (ECMO): Are There Guidelines or Misconceptions

Objectives: To review the clinical practice variations and trends with declaring patients brain dead on ECMO To highlight the need for the development of consensus guidelines to assist clinicians in the accurate diagnosis of brain death in this specific patient populatio

Career development learning in the curriculum: What is an academic’s role?

Career development learning (CDL) is an approach to developing student employability that enables students to reflect on and plan their future careers through engaging in activities outside or within their degree. Building on literature arguing for the benefits of integrating CDL within curriculum, this study examines academics’ perceived roles facilitating CDL. Informed by the principles and processes of Interpretive Phenomenological Analysis (IPA), 55 academics were interviewed from one institution, enabling responses to be examined through a common lens of teaching, policy and governance structures. Findings demonstrate that while some participants broadly understood the value of CDL, the term CDL is not well known. Further, while CDL strategies within teaching contexts occur, they are mostly unplanned or dialogic. This paper presents a taxonomy of current practice, featuring 11 diverse roles for facilitating CDL within curriculum grouped as absent, implicit and explicit approaches. The paper offers recommendations for a university-wide agenda for employability that features CDL strategies embedded across core curricula

Differential proteolytic activation of the Bacillus thuringiensis Cry41Aa parasporin modulates its anticancer effect

Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells) cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made in the N-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60 respectively) which we hypothesize occlude a binding motif

Monte Carlo investigation of the effect of blood volume and oxygen saturation on optical path in reflectance pulse oximetry

Despite the clinical importance of pulse oximetry, the precise nature of the interaction of light with tissue, which underlies the technique, is not yet fully understood. The limitations of the method with regard toits accuracy inconditions of compromised perfusion and/or low blood oxygen saturations are well documented but have only partly been resolved. Results from a static monolayer Monte Carlo modelof optical path and reflectance attwo wavelengths most commonly usedinpulse-oximetry (660 and 940 nm) through skin tissue, containing different volume fractions of blood witharange of oxygen saturations, are presented. Results exhibited differences in mean optical path (MOP) between the two wavelengths, with differences generally increasing with increasing tissue oxygen saturation and decreasing blood volume.As anexample, inatypical sensor configuration, the MOP of red light traveling through skin containing 7.5% blood volume fraction with mean oxygen saturationof60% was 58% higher than that for infrared. The results presented should contribute to further understandingofthe effectofphysiological conditions suchasanemia, ischemia and hypoxemia on the accuracy of pulse oximetry readings

Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

RUNX super-enhancer control through the Notch pathway by Epstein-Barr virus transcription factors regulates B cell growth

In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a −97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (−139 to −250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth