Response to Qian and Colvin : zinc-mediated regulation of the cardiac ryanodine receptor occurs via multiple binding sites

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Modulation of the <i>Neisseria gonorrhoeae </i>drug efflux conduit MtrE

We acknowledge funding through the Wellcome Trust Interdisciplinary Research Funds (grant WT097818MF), the Scottish Universities’ Physics Alliance (SUPA), Tenovus Tayside (grant T16/30) and the Tayside Charitable Trust. O.N.V. has been funded through a BBSRC CASE award (BB/J013072/1).Widespread antibiotic resistance, especially of Gram-negative bacteria, has become a severe concern for human health. Tripartite efflux pumps are one of the major contributors to resistance in Gram-negative pathogens, by efficiently expelling a broad spectrum of antibiotics from the organism. In Neisseria gonorrhoeae, one of the first bacteria for which pan-resistance has been reported, the most expressed efflux complex is MtrCDE. Here we present the electrophysiological characterisation of the outer membrane component MtrE and the membrane fusion protein MtrC, obtained by a combination of planar lipid bilayer recordings and in silico techniques. Our in vitro results show that MtrE can be regulated by periplasmic binding events and that the interaction between MtrE and MtrC is sufficient to stabilize this complex in an open state. In contrast to other efflux conduits, the open complex only displays a slight preference for cations. The maximum conductance we obtain in the in vitro recordings is comparable to that seen in our computational electrophysiology simulations conducted on the MtrE crystal structure, indicating that this state may reflect a physiologically relevant open conformation of MtrE. Our results suggest that the MtrC/E binding interface is an important modulator of MtrE function, which could potentially be targeted by new efflux inhibitors.Publisher PDFPeer reviewe

Structural insights into Ca2+-activated long-range allosteric channel gating of RyR1

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca2+-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 angstrom and a resolution of 4.2 angstrom for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca2+ activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.Strategic Priority Research Program of Chinese Academy of Sciences [XDB08030202]; National Basic Research Program (973 Program); Ministry of Science &amp; Technology of China [2012CB917200, 2014CB910700]; National Natural Science Foundation of China [31270768]; Ministry of Education of China (111 Program China)SCI(E)PubMed中国科技核心期刊(ISTIC)[email protected]; [email protected]

A family of Type VI secretion system effector proteins that form ion-selective pores

This work was supported by the Wellcome Trust (104556/Z/14/Z, Senior Fellowship in Basic Biomedical Science to S.J.C.; 097818/Z/11/B and 109118/Z/15/Z, PhD studentships to University of Dundee), the MRC (MR/K000111X/1, New Investigator Research Grant to S.J.C.) and the Royal Society of Edinburgh (Biomedical Personal Research Fellowship to S.J.P.). We thank Roland Freudl for the gift of anti-OmpA antibody; Adam Ostrowski for construction of strains AO07 and AO08; Gal Horesh, Amy Dorward and Gavin Robertson for expert assistance; the Flow Cytometry and Cell Sorting Facility at the University of Dundee; and the Dundee Imaging Facility (supported by Wellcome Trust [097945/B/11/Z] and MRC [MR/K015869/1]) awards).Type VI secretion systems (T6SSs) are nanomachines widely used by bacteria to deliver toxic effector proteins directly into neighbouring cells. However, the modes of action of many effectors remain unknown. Here we report that Ssp6, an anti-bacterial effector delivered by a T6SS of the opportunistic pathogen Serratia marcescens, is a toxin that forms ion-selective pores. Ssp6 inhibits bacterial growth by causing depolarisation of the inner membrane in intoxicated cells, together with increased outer membrane permeability. Reconstruction of Ssp6 activity in vitro demonstrates that it forms cation-selective pores. A survey of bacterial genomes reveals that genes encoding Ssp6-like effectors are widespread in Enterobacteriaceae and often linked with T6SS genes. We conclude that Ssp6 and similar proteins represent a new family of T6SS-delivered anti-bacterial effectors.Publisher PDFPeer reviewe

The Impact of Entrepreneurship Education in Higher Education: A Systematic Review and Research Agenda

Using a teaching model framework, we systematically review empirical evidence on the impact of entrepreneurship education (EE) in higher education on a range of entrepreneurial outcomes, analyzing 159 published articles from 2004 to 2016. The teaching model framework allows us for the first time to start rigorously examining relationships between pedagogical methods and specific outcomes. Reconfirming past reviews and meta-analyses, we find that EE impact research still predominantly focuses on short-term and subjective outcome measures and tends to severely underdescribe the actual pedagogies being tested. Moreover, we use our review to provide an up-to-date and empirically rooted call for less obvious, yet greatly promising, new or underemphasized directions for future research on the impact of university-based entrepreneurship education. This includes, for example, the use of novel impact indicators related to emotion and mind-set, focus on the impact indicators related to the intention-to-behavior transition, and exploring the reasons for some contradictory findings in impact studies including person-, context-, and pedagogical model-specific moderator

The disenchantment of Southeast Asia: New media and social change post 9/11

Although governments around Southeast Asia bemoan their failing ability to control the flow of news and information in the Internet age, the terrorist attacks on the US and, in particular, those in Bali in October 2002, are likely to provide a fillip for the region’s hard-liners, and underpin surveillance states in the region. As culture becomes a major factor in national security and international relations, the role of the media and communications technology in political change has become ambiguous. The Internet allows the high-tech mobilisation of radical constituencies, and threatens to shake dominant political visions and cultural traditions to the core. Although technology has allowed a greater share of voice for the disillusioned, the dispossessed and the disadvantaged, it is also an effective weapon in the hands of the state. With Southeast Asia’s silent majority prepared to sacrifice gains in democratic pluralism in return for security, the war on ‘terror’ will allow authoritarian governments to reel in many of the gains in freedom of speech only recently won and further alienate the Malay-Islamic communities, driving them into the arms of militant radicals

Intracellular zinc modulates cardiac ryanodine receptor-mediated calcium release

This work was supported by University of St. Andrews, Tenovus Scotland Grant T14/35 (to S. J. P.), British Heart Foundation Grant FS/14/69/31001 (to S. J. P.), a grant from the John and Lucille van Geest Cardiovascular Diseases Research Fund (to the University of Leicester and R. D. R.), and Biotechnology and Biological Sciences Research Council Grant BB/J006467/1 (to A. J. S.).Aberrant Zn2+-homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+-dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nM, Zn2+ is the main activating ligand and the dependency on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single-channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration dependent manner and that physiological levels of Zn2+ elicit Ca2+-release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+-dynamics in cardiomyocytes through modulation of RyR2 gating.Publisher PDFPeer reviewe

Transparency in Health Care: Disclosing Adverse Events to the Public

The topic of transparency has received increasing academic interest in recent years. Transparency can be interpreted as conducting affairs in the open, being subject to public scrutiny, or admitting to problems when they arise. This chapter analyses transparency in disclosing adverse events to the public in Norway. We use the widely publicized Daniel case to show the communication between the regulator and the public, discussing key elements of transparency in the healthcare setting, including the role of media. The Daniel case describes an accidental tonsillectomy characterized by cover-up, failure of the initial regulatory and hospital follow-up, coming to a head when media shone a spotlight on the case. The media coverage caused social amplification of the risk communication resulting in regulatory follow-up having to apply new forms of transparency strategies to rebuild trust in the public. By using the Daniel case as emblematic of Norwegian risk communication strategies in health care, improvements should be made along the lines of direct and adequate information exchange according to patient rights, and efforts to foster open and transparent regulatory and organizational cultures to ensure public trust.publishedVersio