Participation of Glutamate-354 of the CP43 Polypeptide in the Ligation of Manganese and the Binding of Substrate Water in Photosystem II

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn4Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn4Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H218O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S3 state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S1 state Mn-EXAFS spectrum, a slightly altered S2 state multiline EPR signal, a substantially altered S 2-minus-S1 FTIR difference spectrum, and an unusually long lifetime for the S2 state (>10 h) in a substantial fraction of reaction centers. In contrast, the S2 state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S2-minus-S 1 FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with 15N and specific labeling with l-[1-13C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO- group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm -1 in both the S1 and S2 states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S 2 state and that most of these can achieve the S3 state, but no evidence for advancement beyond the S3 state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn4Ca cluster in the S 2 state, the FTIR and H218O exchange data show that the mutation strongly influences other properties of the Mn4Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S1 to S2 transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S3 state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn4Ca cluster in the S1 state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S1 to S 2 transition. The H218O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S 3 state

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Neuroliberalism:Cognition, context, and the geographical bounding of rationality

Focusing on the rise of the behavioural sciences within the design and implementation of public policy, this paper introduces the concept of neuroliberalism and suggests that it could offer a creative context within which to interpret related governmental developments. Understanding neuroliberaism as a system of government that targets the more-than rational aspects of human behaviour, this paper considers the particular contribution that geographical theories of context and spatial representation can make to a critical analysis of this evolving governmental project.authorsversionPeer reviewe

vCJD risk in the Republic of Ireland

BACKGROUND: The Republic of Ireland has the second highest incidence of BSE worldwide. Only a single case of vCJD has been identified to date. METHODS: We estimate the total future number of clinical cases of vCJD using an established mathematical model, and based on infectivity of bovine tissue calculated from UK data and on the relative exposure to BSE contaminated meat. RESULTS: We estimate 1 future clinical case (95% CI 0 – 15) of vCJD in the Republic of Ireland. Irish exposure is from BSE infected indigenous beef products and from imported UK beef products. Additionally, 2.5% of the Irish population was exposed to UK beef through residing in the UK during the 'at-risk' period. The relative proportion of risk attributable to each of these three exposures individually is 2:2:1 respectively. CONCLUSIONS: The low numbers of future vCJD cases estimated in this study is reassuring for the Irish population and for other countries with a similar level of BSE exposure

Cross-National Analysis of the Associations among Mental Disorders and Suicidal Behavior: Findings from the WHO World Mental Health Surveys

Using data from over 100,000 individuals in 21 countries participating in the WHO World Mental Health Surveys, Matthew Nock and colleagues investigate which mental health disorders increase the odds of experiencing suicidal thoughts and actual suicide attempts, and how these relationships differ across developed and developing countries

Disclosure of physical, emotional and sexual child abuse, help-seeking and access to abuse response services in two South African Provinces

Physical, emotional and sexual child abuse are major problems in South Africa. This study investigates whether children know about post-abuse services, if they disclose and seek services, and what the outcomes of help-seeking behaviour are. It also examines factors associated with request and receipt of services. Confidential self-report questionnaires were completed by adolescents in South Africa as part of a communitybased study of children aged 10-17 (n=3515) in two provinces. Child abuse, disclosure and outcomes of help-seeking were measured using internationally recognized measures. Prevalence of frequent (>weekly) physical abuse was 7.4%, frequent emotional abuse 12.4%, and lifetime contact sexual abuse 9.0%. 98.6% of children could name one suitable confidante or formal service for abuse disclosure, but only 20.1% of abuse victims disclosed. Of those, 72% received help. Most common confidantes were caregivers and teachers. Of all abuse victims, 85.6% did not receive help due to non-disclosure or inactivity of services, and 14.4% received help: 4.9% from formal health or social services and 7.1% through community vigilante action. Emotional abuse, sexual abuse and female gender were associated with higher odds of help-seeking. While children in South Africa showed high knowledge of available services, access to formal services among abused children was low and not all those requesting services received them. Notably fewer children received help from formal services than through community vigilante action. Urgent action is needed to improve service access for child abuse victims

FTIR Difference Spectroscopy Studies of Residue Roles at the Mn4Ca Cluster and the Hydrogen Bonding Network in Photosystem II

The photosynthetic protein, Photosystem II (PSII) found in both plants and cyanobacteria is the center for the intricate processes of water oxidation and oxygen evolution. At the oxygen evolving complex (OEC) is a four manganese and calcium (Mn4Ca) cluster and surrounding carboxylate and histidine residues, which help control the active site environment and oxidizing water molecules through a series of electron transfer steps. The OEC accrues oxidizing equivalents in several steps during the catalytic cycle called storage states, or S-states, resulting in protons, electrons, and dioxygen, which is then released and carried out from the active site through a channel within the protein. Identifying the residues involved in this procedure as well as those which stabilize the catalytic cycle through a series of hydrogen bonds is important in understanding the mechanism as a whole. Spectroscopic methods of analysis such as Fourier transform infrared (FTIR) difference spectroscopy can illustrate the dynamic nature of bond creation/destruction that occurs in structural rearrangement. FTIR spectroscopy analysis reproducibly shows many vibrational modes for each S-state throughout the cycle, indicating an arranged routine in water oxidation, are best viewed in the midfrequency region for amide I/II bending, symmetric/asymmetric carboxylate stretching, and carbonyl of carboxylic acid stretching. Changes in vibrational modes, shown as positive or negative peaks, indicate residue participation either at the active site or possibly a water or proton channel.Data have been collected for two ligands to the Mn4Ca cluster, CP43-Glu354 and D1-Glu333 characterizing the former as a ligand which does not change its coordination mode during the S1 to S2 transition but does show evidence that ligation takes place at Mn3 and Mn4 ions. The latter plays an integral role in structure reinforcement as well as dynamic interactions, though the specific activity is not yet clear. Two different channels, an oxygen channel (large) and water or proton egress channel (broad) were observed. Of the large, CP43-Glu354 and D1-Glu329 were included. Of the broad, D1-Glu65, D2-Glu312, D1-Glu333, D2-Glu323, and D2-Lys317 were observed. For the large channel, CP43-Glu354 gave evidence of controlling a hydrogen-bonding network but not directly engaging unlike the D1-Glu329 which does. For the broad, only D1-Glu65 and D2-Glu312 showed clear signs of participating in the network. D1-Glu333 and D2-Lys317 showed evidence of important interaction in structure stability, a likely situation considering proximity to a chloride ion. This study shows the complex and sensitive nature for activity throughout PSII and not just in the OEC during the catalytic cycle

Network of hydrogen bonds near the oxygen-evolving Mn 4 CaO 5 cluster of Photosystem II probed with FTIR difference spectroscopy

We previously provided experimental evidence that an extensive network of hydrogen bonds exists near the oxygen-evolving Mn4CaO5 cluster in photosystem II and that elements of this network form part of a dominant proton-egress pathway leading from the Mn4CaO5 cluster to the thylakoid lumen. The evidence was based on (i) the elimination of the same ν(C=O) mode of a protonated carboxylate group in the S 2-minus-S1 FTIR difference spectrum of wild-type PSII core complexes from the cyanobacterium Synechocystis sp. PCC 6803 by the mutations D1-E65A, D2-E312A, and D1-E329Q and (ii) the substantial decrease in the efficiency of the S3 to S0 transition caused by the mutations D1-D61A, D1-E65A, and D2-E312A. The eliminated ν(C=O) mode corresponds to an unidentified carboxylate group whose pKa value decreases in response to the increased charge that develops on the Mn 4CaO5 cluster during the S1 to S2 transition. In the current study, we have extended our work to include the ν(C=O) regions of other Sn+1-minus-Sn FTIR difference spectra and to additional mutations of residues inferred to participate in networks of hydrogen bonds near the Mn4CaO5 cluster or leading from the Mn4CaO5 cluster to the thylakoid lumen. Our data suggest that a different carboxylate group has its pKa value increased during the S2 to S3 transition and that a third carboxylate group experiences a change in its environment during the S 0 to S1 transition. The pKa values that shift during the S1 to S2 and S2 to S3 transitions appear to be restored during the S3 to S0 transition. The D1-R334A mutation decreases or eliminates the same ν(C=O) modes from the S2-minus-S1 and S3-minus-S 2 spectra as mutations D1-E65A, D2-E312A, and D1-E329Q and substantially decreases the efficiency of the S3 to S0 transition. We conclude that D1-R334 participates in the same dominant proton-egress pathway that was identified in our previous study. The D1-Q165E mutation leaves the ν(C=O) region of the S2-minus-S1 FTIR difference spectrum intact, but it eliminates a mode from this region of the S3-minus-S2 spectrum. We conclude that D1-Q165 participates in an extensive network of hydrogen bonds that that extends across the Mn4CaO5 cluster to the D1-E65/D2-E312 dyad and that includes D1-E329 and several water molecules including the W2 and W3 water ligands of the Mn4CaO5 cluster's dangling MnA4 and Ca ions, respectively. The D2-E307Q, D2-D308N, D2-E310Q, and D2-E323Q mutations alter the ν(C=O) regions of none of the FTIR difference spectra. We conclude that these four residues are located far from the three unidentified carboxylate groups that give rise to the ν(C=O) features observed in the FTIR difference spectra

Evidence from FTIR Difference Spectroscopy of an Extensive Network of Hydrogen Bonds near the Oxygen-Evolving Mn4Ca Cluster of Photosystem II Involving D1-Glu65, D2-Glu312, and D1-Glu329

Analyses of the refined X-ray crystallographic structures of photosystem II (PSII) at 2.9-3.5 Å have revealed the presence of possible channels for the removal of protons from the catalytic Mn4Ca cluster during the water-splitting reaction. As an initia