The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

Type II DNA topoisomerases (topos) are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD) of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity.We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity.In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs

Resolution of LPS-induced airway inflammation and goblet cell hyperplasia is independent of IL-18

BACKGROUND: The resolution of inflammatory responses in the lung has not been described in detail and the role of specific cytokines influencing the resolution process is largely unknown. METHODS: The present study was designed to describe the resolution of inflammation from 3 h through 90 d following an acute injury by a single intratracheal instillation of F344/N rats with LPS. We documented the inflammatory cell types and cytokines found in the bronchoalveolar lavage fluid (BALF), and epithelial changes in the axial airway and investigated whether IL-18 may play a role in the resolution process by reducing its levels with anti-IL-18 antibodies. RESULTS: Three major stages of inflammation and resolution were observed in the BALF during the resolution. The first stage was characterized by PMNs that increased over 3 h to 1 d and decreased to background levels by d 6–8. The second stage of inflammation was characterized by macrophage influx reaching maximum numbers at d 6 and decreasing to background levels by d 40. A third stage of inflammation was observed for lymphocytes which were elevated over d 3–6. Interestingly, IL-18 and IL-9 levels in the BALF showed a cyclic pattern with peak levels at d 4, 8, and 16 while decreasing to background levels at d 1–2, 6, and 12. Depletion of IL-18 caused decreased PMN numbers at d 2, but no changes in inflammatory cell number or type at later time points. CONCLUSION: These data suggest that IL-18 plays a role in enhancing the LPS-induced neutrophilic inflammation of the lung, but does not affect the resolution of inflammation

Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming

Human induced pluripotent stem cells (iPSCs) provide a valuable model for regenerative medicine and human disease research. To date, however, the reprogramming efficiency of human adult cells is still low. Recent studies have revealed that cell cycle is a key parameter driving epigenetic reprogramming to pluripotency. As is well known, retroviruses such as the Moloney murine leukemia virus (MoMLV) require cell division to integrate into the host genome and replicate, whereas the target primary cells for reprogramming are a mixture of several cell types with different cell cycle rhythms. Whether cell cycle synchronization has potential effect on retrovirus induced reprogramming has not been detailed. In this study, utilizing transient serum starvation induced synchronization, we demonstrated that starvation generated a reversible cell cycle arrest and synchronously progressed through G2/M phase after release, substantially improving retroviral infection efficiency. Interestingly, synchronized human dermal fibroblasts (HDF) and adipose stem cells (ASC) exhibited more homogenous epithelial morphology than normal FBS control after infection, and the expression of epithelial markers such as E-cadherin and Epcam were strongly activated. Futhermore, synchronization treatment ultimately improved Nanog positive clones, achieved a 15–20 fold increase. These results suggested that cell cycle synchronization promotes the mesenchymal to epithelial transition (MET) and facilitates retrovirus mediated reprogramming. Our study, utilization of serum starvation rather than additional chemicals, provide a new insight into cell cycle regulation and induced reprogramming of human cells

Lazarus1, a DUF300 Protein, Contributes to Programmed Cell Death Associated with Arabidopsis acd11 and the Hypersensitive Response

Programmed cell death (PCD) is a necessary part of the life of multi-cellular organisms. A type of plant PCD is the defensive hypersensitive response (HR) elicited via recognition of a pathogen by host resistance (R) proteins. The lethal, recessive accelerated cell death 11 (acd11) mutant exhibits HR-like accelerated cell death, and cell death execution in acd11 shares genetic requirements for HR execution triggered by one subclass of R proteins

Integrated vector management for malaria control

Integrated vector management (IVM) is defined as "a rational decision-making process for the optimal use of resources for vector control" and includes five key elements: 1) evidence-based decision-making, 2) integrated approaches 3), collaboration within the health sector and with other sectors, 4) advocacy, social mobilization, and legislation, and 5) capacity-building. In 2004, the WHO adopted IVM globally for the control of all vector-borne diseases. Important recent progress has been made in developing and promoting IVM for national malaria control programmes in Africa at a time when successful malaria control programmes are scaling-up with insecticide-treated nets (ITN) and/or indoor residual spraying (IRS) coverage. While interventions using only ITNs and/or IRS successfully reduce transmission intensity and the burden of malaria in many situations, it is not clear if these interventions alone will achieve those critical low levels that result in malaria elimination. Despite the successful employment of comprehensive integrated malaria control programmes, further strengthening of vector control components through IVM is relevant, especially during the "end-game" where control is successful and further efforts are required to go from low transmission situations to sustained local and country-wide malaria elimination. To meet this need and to ensure sustainability of control efforts, malaria control programmes should strengthen their capacity to use data for decision-making with respect to evaluation of current vector control programmes, employment of additional vector control tools in conjunction with ITN/IRS tactics, case-detection and treatment strategies, and determine how much and what types of vector control and interdisciplinary input are required to achieve malaria elimination. Similarly, on a global scale, there is a need for continued research to identify and evaluate new tools for vector control that can be integrated with existing biomedical strategies within national malaria control programmes. This review provides an overview of how IVM programmes are being implemented, and provides recommendations for further development of IVM to meet the goals of national malaria control programmes in Africa

Signal processing and transduction in plant cells: the end of the beginning?

Plants have a very different lifestyle to animals, and one might expect that unique molecules and processes would underpin plant-cell signal transduction. But, with a few notable exceptions, the list is remarkably familiar and could have been constructed from animal studies. Wherein, then, does lifestyle specificity emerge

Prolonged immune alteration following resolution of acute inflammation in humans.

Acute inflammation is an immediate response to infection and injury characterised by the influx of granulocytes followed by phagocytosing mononuclear phagocytes. Provided the antigen is cleared and the immune system of the host is fully functional, the acute inflammatory response will resolve. Until now it is considered that resolution then leads back to homeostasis, the physiological state tissues experienced before inflammation occurred. Using a human model of acute inflammation driven by intradermal UV killed Escherichia coli, we found that bacteria and granulocyte clearance as well as pro-inflammatory cytokine catabolism occurred by 72h. However, following a lag phase of about 4 days there was an increase in numbers of memory T cells and CD163+ macrophage at the post-resolution site up to day 17 as well as increased biosynthesis of cyclooxygenase-derived prostanoids and DHA-derived D series resolvins. Inhibiting post-resolution prostanoids using naproxen showed that numbers of tissue memory CD4 cells were under the endogenous control of PGE2, which exerts its suppressive effects on T cell proliferation via the EP4 receptor. In addition, we re-challenged the post-resolution site with a second injection of E. coli, which when compared to saline controls resulted in primarily a macrophage-driven response with comparatively fewer PMNs; the macrophage-dominated response was reversed by cyclooxygenase inhibition. Re-challenge experiments were also carried out in mice where we obtained similar results as in humans. Therefore, we report that acute inflammatory responses in both humans and rodents do not revert back to homeostasis, but trigger a hitherto unappreciated sequence of immunological events that dictate subsequent immune response to infection.Wellcome Trust Senior Research Fellowship (Grant number: WT087520), Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant number: 107613/Z/15/Z) and the Barts Charity (Grant number: MGU0343)