Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage

The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M(r) complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex

Revisiting Clickers: In-Class Questions Followed by At-Home Reflections Are Associated with Higher Student Performance on Related Exam Questions

Clicker questions are a commonly used active learning technique that stimulates student interactions to help advance understanding of key concepts. Clicker questions are often administered with an initial vote, peer discussion, and a second vote, followed by broader classroom explanation. While clickers can promote learning, some studies have questioned whether students maintain this performance on later exams, highlighting the need to further understand how student answer patterns relate to their understanding of the material and to identify ways for clickers to benefit a broader range of students. Systematic requizzing of concepts during at-home assignments represents a promising mechanism to improve student learning. Thus, we paired clicker questions with at-home follow-up reflections to help students articulate and synthesize their understandings. This pairing of clickers with homework allowed us to decipher how student answer patterns related to their underlying conceptions and to determine if revisiting concepts provided additional benefits. We found that students answering both clicker votes correctly performed better on isomorphic exam questions and that students who corrected their answers after the first vote did not show better homework or exam performance than students who maintained an incorrect answer across both votes. Furthermore, completing the followup homework assignment modestly boosted exam question performance. Our data suggest that longer-term benefits of clickers and associated homework may stem from students having repeated opportunities to retrieve, refine, and reinforce emerging conceptions

Regulation of neutrophilic inflammation by hypoxic signalling pathways

Neutrophils are essential for effective innate immunity. Conversely, inappropriate or excessive neutrophil activation can result in damaging inflammation. This damage is implicated in the pathogenesis of a number of respiratory diseases including acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD) which are also both frequently complicated by hypoxia. Cells sense and respond to hypoxia through the activity of the transcription factor HIF (hypoxia inducible factor) and its regulatory hydroxylases, the prolyl hydroxylase domain enzymes (PHDs) 1- 3. In the presence of oxygen, PHDs hydroxylate HIF, preventing the HIF mediated transcriptional response. Close links exist between the pathways which regulate hypoxic and inflammatory responses. Our group has previously found that in mouse models of infection, acute hypoxia leads to increased sickness and that this is driven by neutrophilic inflammation. I have used a murine model of Lipopolysaccharide (LPS) -induced acute lung injury, characterised by neutrophil influx, to investigate how exposure to hypoxia alters lung inflammation. Using high-resolution mass spectrometry, I have defined the proteome of the inflammatory lung neutrophil. I have shown that hypoxia results in a distinct proteomic signature in inflammatory neutrophils. Hypoxia drives lung neutrophilic inflammation through increased neutrophil degranulation and upregulation of inflammatory receptors. I have also identified key metabolic alterations in hypoxic neutrophils. The hypoxic lung represents a low glucose, high protein environment and neutrophils adapt to exploit this. I have shown that neutrophils can scavenge proteins from their extracellular environment, catabolise these proteins in the lysosome and utilise the breakdown products for metabolism. These processes are upregulated in hypoxic lung neutrophils which show increased lysosomal protein expression, increased protein uptake and increased glutaminolysis. Utilising heavy labelled protein extracts, I have traced breakdown products from scavenged proteins into central carbon metabolism, demonstrating that extracellular protein can fuel neutrophilic inflammation. Finally, I have investigated the role of the prolyl hydroxylase PHD1 in regulating neutrophilic inflammation. Using a neutrophil specific PHD1 knockout mouse line, I have identified a specific role for PHD1 in regulating neutrophil metabolism and survival. I have found that the micro-environment, particularly oxygen availability, determines the impact of PHD1 loss with consequences for inflammation resolution in vivo. In summary, hypoxia is a key regulator of neutrophil function and is associated with increased neutrophilic inflammation. Utilising a proteomic approach, I have identified the mechanisms which drive the hyperinflammatory phenotype including the ability of neutrophils to scavenge proteins from the environment to fuel inflammation. I have also shown that PHD1, a key component of the hypoxic signalling pathway, may regulate these functions. A more complete understanding of these mechanisms will help to identify therapeutic targets for treatment of neutrophilic inflammation in the lung

IL4Rα signaling abrogates hypoxic neutrophil survival and limits acute lung injury responses <i>in vivo</i>

Rationale: Acute respiratory distress syndrome is defined by the presence of systemic hypoxia and consequent on disordered neutrophilic inflammation. Local mechanisms limiting the duration and magnitude of this neutrophilic response remain poorly understood.  Objectives: To test the hypothesis that during acute lung inflammation tissue production of proresolution type 2 cytokines (IL-4 and IL-13) dampens the proinflammatory effects of hypoxia through suppression of HIF-1a (hypoxia-inducible factor-1a)mediated neutrophil adaptation, resulting in resolution of lung injury.  Methods: Neutrophil activation of IL4Ra (IL-4 receptor a) signaling pathways was explored ex vivo in human acute respiratory distress syndrome patient samples, in vitro after the culture of human peripheral blood neutrophils with recombinant IL-4 under conditions of hypoxia, and in vivo through the study of IL4Ra-deficient neutrophils in competitive chimera models and wild-type mice treated with IL-4.  Measurements and Main Results: IL-4 was elevated in human BAL from patients with acute respiratory distress syndrome, and its receptor was identified on patient blood neutrophils. Treatment of human neutrophils with IL-4 suppressed HIF-1a-dependent hypoxic survival and limited proinflammatory transcriptional responses. Increased neutrophil apoptosis in hypoxia, also observed with IL-13, required active STAT signaling, and was dependent on expression of the oxygen-sensing prolyl hydroxylase PHD2. In vivo, IL-4Ra-deficient neutrophils had a survival advantage within a hypoxic inflamed niche; in contrast, inflamed lung treatment with IL-4 accelerated resolution through increased neutrophil apoptosis.  Conclusions: We describe an important interaction whereby IL4Ra-dependent type 2 cytokine signaling can directly inhibit hypoxic neutrophil survival in tissues and promote resolution of neutrophil-mediated acute lung injury

Hypoxia drives murine neutrophil protein scavenging to maintain central carbon metabolism

Limiting dysfunctional neutrophilic inflammation while preserving effective immunity requires a better understanding of the processes that dictate neutrophil function in the tissues. Quantitative mass-spectrometry identified how inflammatory murine neutrophils regulated expression of cell surface receptors, signal transduction networks, and metabolic machinery to shape neutrophil phenotypes in response to hypoxia. Through the tracing of labeled amino acids into metabolic enzymes, proinflammatory mediators, and granule proteins, we demonstrated that ongoing protein synthesis shapes the neutrophil proteome. To maintain energy supplies in the tissues, neutrophils consumed extracellular proteins to fuel central carbon metabolism. The physiological stresses of hypoxia and hypoglycemia, characteristic of inflamed tissues, promoted this extracellular protein scavenging with activation of the lysosomal compartment, further driving exploitation of the protein-rich inflammatory milieu. This study provides a comprehensive map of neutrophil proteomes, analysis of which has led to the identification of active catabolic and anabolic pathways that enable neutrophils to sustain synthetic and effector functions in the tissues

A feasibility study of a psycho-educational support intervention for men with prostate cancer on active surveillance.

Background: PROACTIVE is a psycho-educational support intervention for prostate cancer patients managed on Active Surveillance. PROACTIVE is comprised of two interdependent components: group workshops and internet delivered information modules. Aims: We conducted a feasibility study to determine the practicality of delivering PROACTIVE at two prostate cancer centres. Methods: The feasibility study was a mixed methods randomized parallel-group exploratory trial. Participants were randomised using a ratio of 3:1 PROACTIVE group to treatment as usual. Qualitative semi-structured interviews and quantitative measures were completed at baseline, intervention completion (week 6), and at 6-months follow-up. Interview transcripts were analysed thematically using Framework analysis. Descriptive statistics were used to examine recruitment and retention rates, and changing trends in outcome measures. Results: Most aspects of the research design and PROACTIVE intervention were acceptable to those participating in the study. In particular participants valued the opportunity to share and discuss experiences with other prostate cancer patients on Active Surveillance, and receive detailed authoritative information. However, three issues were identified: 1. a low response rate (13 participants recruited, response rate 16%) 2. low utilisation of internet delivered information modules 3. self-perceived low levels of anxiety amongst participants with the majority perceiving their cancer as not impacting on their day-to-day life or causing anxiety. Conclusions: Due to these significant research design issues it is not recommended PROACTIVE be evaluated in a large scale randomised controlled trial. Further research is required to explore the impact of Active Surveillance on anxiety amongst men with localized prostate cancer managed by Active Surveillance

Mitigating the impact of Bats in historic churches: The response of Natterer's Bats Myotis nattereri to artificial roosts and deterrence

© 2016 Zeale et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Bats frequently roost in historic churches, and these colonies are of considerable conservation value. Inside churches, bat droppings and urine can cause damage to the historic fabric of the building and to items of cultural significance. In extreme cases, large quantities of droppings can restrict the use of a church for worship and/or other community functions. In the United Kingdom, bats and their roosts are protected by law, and striking a balance between conserving the natural and cultural heritage can be a significant challenge. We investigated mitigation strategies that could be employed in churches and other historic buildings to alleviate problems caused by bats without adversely affecting their welfare or conservation status. We used a combination of artificial roost provision and deterrence at churches in Norfolk, England, where significant maternity colonies of Natterer's bats Myotis nattereri damage church features. Radio-tracking data and population modelling showed that excluding M. nattereri from churches is likely to have a negative impact on their welfare and conservation status, but that judicious use of deterrents, especially high intensity ultrasound, can mitigate problems caused by bats. We show that deterrence can be used to move bats humanely from specific roosting sites within a church and limit the spread of droppings and urine so that problems to congregations and damage to cultural heritage can be much reduced. In addition, construction of bespoke roost spaces within churches can allow bats to continue to roost within the fabric of the building without flying in the church interior. We highlight that deterrence has the potential to cause serious harm toM. nattereri populations if not used judiciously, and so the effects of deterrents will need careful monitoring, and their use needs strict regulation