Restoration of marine ecosystems: Understanding possible futures for optimal outcomes.

Accelerating declines in the extent, quality and functioning of the world's marine ecosystems have generated an upsurge in focus on practical solutions, with ecosystem restoration becoming an increasingly attractive mitigation strategy for systems as diverse as coral reefs, mangroves and tidal flats. While restoration is popular because it promises positive outcomes and a return to something approaching unimpacted condition and functioning, it involves substantial public and private investment, both for the initial restoration activity and for on-going maintenance of the restored asset. This investment often affords one big chance to get things right before irretrievable damage is done. As a result, precise, well considered and accountable decision-making is needed to determine the specific focus for restoration, the scale of restoration, the location for deploying restoration activities, and indeed whether or not restoration is necessary or even possible. We explore the environmental/ecological considerations and constraints governing optimal decisions about the nature, location and prioritisation of restoration activities in marine ecosystems, and in particular the constraints on achieving understanding of possible futures and the likelihood of achieving them. We conclude that action must be informed by a context-specific understanding of the historical situation, the current situation, the constraints on change, the range of potential outcome scenarios, and the potential futures envisioned.

Identification and validation of the mode of action of the chalcone anti-mycobacterial compounds.

OBJECTIVES: The search for new TB drugs has become one of the great challenges for modern medicinal chemistry. An improvement in the outcomes of TB chemotherapy can be achieved by the development of new, shorter, cheap, safe and effective anti-TB regimens. METHODS: Chalcones (1a-1o) were synthesized and evaluated for their antimycobacterial activity against Mycobacterium bovis BCG using growth inhibition assays. Compound 1a was selected as a 'hit' compound. The mode of action of compound 1a, was identified by mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs) analysis using thin layer chromatography. Dose dependent experiments were conducted by over-expressing components of FAS-II in M. bovis BCG to confirm the target. Ligand binding using intrinsic tryptophan assay and molecular docking were used to further validate the target. RESULTS: MAMEs and FAMEs analysis showed dose-dependent reduction of MAMEs with the overall abundance of FAMEs suggesting that compound 1a targets mycolic acid biosynthesis. Direct binding of 1a to InhA was observed using an intrinsic tryptophan fluorescence binding assay, and a 2-fold IC50 shift was observed with an InhA overexpressing strain confirming InhA as the cellular target. CONCLUSION: The chalcone 1a exhibits potent antimycobacterial activity, displays a good safety profile and is a direct inhibitor of InhA, a key component in mycolic acid synthesis, validating this series for further anti-TB drug development.

Our Environmental Value Orientations Influence How We Respond to Climate Change.

People variably respond to global change in their beliefs, behaviors, and grief (associated with losses incurred). People that are less likely to believe in climate change, adopt pro-environmental behaviors, or report ecological grief are assumed to have different psycho-cultural orientations, and do not perceive changes in environmental condition or any impact upon themselves. We test these assumptions within the context of the Great Barrier Reef (GBR), a region currently experiencing significant climate change impacts in the form of coral reef bleaching and increasingly severe cyclones. We develop knowledge of environmental cultural services with the Environmental Schwartz Value Survey (ESVS) into four human value orientations that can explain individuals' environmental beliefs and behaviors: biospheric (i.e., concern for environment), altruistic (i.e., concern for others, and intrinsic values), egoistic (i.e., concern for personal resources) and hedonic values (i.e., concern for pleasure, comfort, esthetic, and spirituality). Using face-to-face quantitative survey techniques, where 1,934 residents were asked to agree or disagree with a range of statements on a scale of 1-10, we investigate people's (i) environmental values and value orientations, (ii) perceptions of environmental condition, and (iii) perceptions of impact on self. We show how they relate to the following climate change responses; (i) beliefs at a global and local scale, (ii) participation in pro-environmental behaviors, and (iii) levels of grief associated with ecological change, as measured by respective single survey questions. Results suggest that biospheric and altruistic values influenced all climate change responses. Egoistic values were only influential on grief responses. Perception of environmental change was important in influencing beliefs and grief, and perceptions of impact on self were only important in influencing beliefs. These results suggest that environmental managers could use people's environmental value orientations to more effectively influence climate change responses toward environmental stewardship and sustainability. Communications that target or encourage altruism (through understanding and empathy), biospherism (through information on climate change impacts on the environment), and egoism (through emphasizing the benefits, health and wellbeing derived from a natural resource in good condition), could work.

A hyperspectral fluorescence lifetime probe for skin cancer diagnosis.

The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440 nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2 h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.

Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging.

We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.

Amyloid beta-peptide(1-42) and hydrogen peroxide-induced toxicity are mediated by TRPM2 in rat primary striatal cultures.

Amyloid beta-peptide (Abeta) is the main component of senile plaques which characterize Alzheimer's disease and may induce neuronal death through mechanisms which include oxidative stress. To date, the signalling pathways linking oxidant stress, a component of several neurodegenerative diseases, to cell death in the CNS are poorly understood. Melastatin-like transient receptor potential 2 (TRPM2) is a Ca(2+)-permeant non-selective cation channel, which responds to increases in oxidative stress levels in the cell and is activated by oxidants such as hydrogen peroxide. We demonstrate here that Abeta and hydrogen peroxide both induce death in cultured rat striatal cells which express TRPM2 endogenously. Transfection with a splice variant that acts as a dominant negative blocker of TRPM2 function (TRPM2-S) inhibited both hydrogen peroxide- and Abeta-induced increases in intracellular-free Ca(2+) and cell death. Functional inhibition of TRPM2 activation by the poly(ADP-ribose)polymerase inhibitor SB-750139, a modulator of intracellular pathways activating TRPM2, attenuated hydrogen peroxide- and Abeta-induced cell death. Furthermore, a small interfering RNA which targets TRPM2, reduced TRPM2 mRNA levels and the toxicity induced by hydrogen peroxide and Abeta. These data demonstrate that activation of TRPM2, functionally expressed in primary cultures of rat striatum, contributes to Abeta- and oxidative stress-induced striatal cell death.

Flufenamic acid is a pH-dependent antagonist of TRPM2 channels.

Like a number of other TRP channels, TRPM2 is a Ca(2+)-permeable non-selective cation channel, the activity of which is regulated by intracellular and extracellular Ca(2+). A unique feature of TRPM2 is its activation by ADP-ribose and chemical species that arise during oxidative stress, for example, NAD(+) and H(2)O(2). These properties have lead to proposals that this channel may play a role in the cell death produced by pathological redox states. The lack of known antagonists of this channel have made these hypotheses difficult to test. Here, we demonstrate, using patch clamp electrophysiology, that the non-steroidal anti-inflammatory compound flufenamic acid (FFA) inhibits recombinant human TRPM2 (hTRPM2) as well as currents activated by intracellular ADP-ribose in the CRI-G1 rat insulinoma cell line. All concentrations tested in a range from 50 to 1000 microM produced complete inhibition of the TRPM2-mediated current. Following FFA removal, a small (typically 10-15%) component of current was rapidly recovered (time constant approximately 3 s), considerably longer periods in the absence of FFA produced no further current recovery. Reapplication of FFA re-antagonised the recovered current and subsequent FFA washout produced recovery of only a small percentage of the reblocked current. Decreasing extracellular pH accelerated FFA inhibition of TRPM2. Additional experiments indicated hTRPM2 activation was required for FFA antagonism to occur and that the generation of irreversible antagonism was preceded by a reversible component of block. FFA inhibition could not be induced by intracellular application of FFA. ADP-ribose activated currents in the rat insulinoma cell line CRI-G1 were also antagonised by FFA with concentration- and pH-dependent kinetics. In contrast to the observations made with hTRPM2, antagonism of ADP-ribose activated currents in CRI-G1 cells could be fully reversed following FFA removal. These experiments suggest that FFA may be a useful tool antagonist for studies of TRPM2 function.

From DNA structure to gene expression: mediators of nuclear compartmentalization and dynamics.

Eukaryotic genomes are functionally compartmentalized into chromatin domains by their attachment to a supporting structure that has traditionally been termed the nuclear matrix. Present evidence indicates the dynamics of this entity, which requires particular properties of the elements that mediate this kind of interaction. Above all, this is enabled by the so-called 'mass binding phenomenon' by which scaffold/matrix-attachment regions (S/MARs) reversibly associate with ubiquitous factors. Recent investigations and novel techniques have shown that these contacts can be altered by modulators as well as by specific interactions with the components of enhancers and locus control regions.

Computational and in vitro analysis of destabilized DNA regions in the interferon gene cluster: potential of predicting functional gene domains.

Recent evidence adds support to a traditional concept according to which the eukaryotic nucleus is organized into functional domains by scaffold or matrix attachment regions (S/MARs). These regions have previously been predicted to have a high potential for stress-induced duplex destabilization (SIDD). Here we report the parallel results of binding (reassociation) and computational SIDD analyses for regions within the human interferon gene cluster on the short arm of chromosome 9 (9p22). To verify and further refine the biomathematical methods, we focus on a 10 kb region in the cluster with the pseudogene IFNWP18 and the interferon alpha genes IFNA10 and IFNA7. In a series of S/MAR binding assays, we investigate the promoter and termination regions and additional attachment sequences that were detected in the SIDD profile. The promoters of the IFNA10 and the IFNA7 genes have a moderate approximately 20% binding affinity to the nuclear matrix; the termination sequences show stronger association (70-80%) under our standardized conditions. No comparable destabilized elements were detected flanking the IFNWP18 pseudogene, suggesting that selective pressure acts on the physicochemical properties detected here. In extended, noncoding regions a striking periodicity is found of rather restricted SIDD minima with scaffold binding potential. By various criteria, the underlying sequences represent a new class of S/MARs, thought to be involved in a higher level organization of the genome. Together, these data emphasize the relevance of SIDD calculations as a valid approach for the localization of structural, regulatory, and coding regions in the eukaryotic genome.

Vanilloid and TRP channels: a family of lipid-gated cation channels.

The emergence of the TRP (C) and vanilloid (TRPV) receptor family of Ca(2+) permeable channels has started to provide molecular focus to a linked group of ion channels whose common feature is activation primarily by intracellular ligands. These channels have a central role in Ca(2+) homeostasis in virtually all cells and in particular those that lack voltage-gated Ca(2+) channels. We will discuss recent work that is more precisely defining both molecular form and physiological function of this important group of Ca(2+) permeable channels with particular focus on the intracellular ligands that gate and modulate channel activity.

SB-334867-A antagonises orexin mediated excitation in the locus coeruleus.

Electrophysiological recordings from identified noradrenergic locus coeruleus (LC) neurones in rat brain slices have revealed that the orexins can cause direct and reversible depolarisation of the postsynaptic membrane. Whilst it is known that the membrane depolarisation produced by orexin-A can triple the firing rate of spontaneously active LC neurones, quantitative pharmacological analysis that determines the receptor subtype(s) mediating the orexinergic response has not yet been performed. Here we demonstrate that the effects of orexin-A are five-fold more potent than orexin-B on LC neuronal excitability. We show further that the orexin receptor antagonist SB-334867-A inhibits the effects of both agonists with pK(B) values similar to those calculated for human OX1 receptors expressed in CHO cells. Finally, we found no evidence for tonic activation of OX1 receptors in LC noradrenergic neurones despite electron microscopic evidence that orexin terminals directly contact these neurones. These data demonstrate that SB-334867-A is a useful tool compound with which to study the physiology of OX1 receptors.

Germ-line transcripts of the immunoglobulin lambda J-C clusters in the mouse: characterization of the initiation sites and regulatory elements.

Transcription of unrearranged immunoglobulin gene segments strongly correlates with their accessibility to the V(D)J recombination machinery. The regulatory mechanisms governing this germ-line transcription are still poorly defined. In order to identify new regulatory elements, we first carried out a detailed characterization of the transcription initiation sites for the J-C germ-line transcripts, using rapid amplification of 5' cDNA ends, assisted by a template switching mechanism at the 5'-end of the RNA. Transcripts were observed that initiated heterogeneously, starting up to 293 (lambda1), 116 bp (lambda2) and 79 bp (lambda3) upstream from the respective Jlambda gene segment. Additional RT-PCR analysis revealed the existence of germ-line transcripts of lambda and also of kappa that initiate even more upstream of these transcription initiation sites, although their frequencies were low. Promoter activity was detected in vitro 5' of Jlambda2, with the minimal promoter activity mapping to the region between positions -35 and -120. In addition, computer analysis allowed the prediction of a nuclear scaffold/matrix attachment (S/MAR) region between the two J-C gene clusters at each hemi-locus. This region between the lambda1/lambda3 clusters binds to the nuclear matrix in vitro, and J-C lambda1 germ-line transcription initiates a short distance downstream from this S/MAR element.

AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs.

Interstrand cross-links at T(A/T)4A sites in cellular DNA are associated with hypercytotoxicity of an anticancer drug, bizelesin. Here we evaluated whether these lethal effects reflect targeting critical genomic regions. An in silico analysis of human sequences showed that T(A/T)4A motifs are on average scarce and scattered. However, significantly higher local motif densities were identified in distinct minisatellite regions (200-1000 base pairs of approximately 85-100% AT), herein referred to as "AT islands." Experimentally detected bizelesin lesions agree with these in silico predictions. Actual bizelesin adducts clustered within the model AT island naked DNA, whereas motif-poor sequences were only sparsely adducted. In cancer cells, bizelesin produced high levels of lesions (approximately 4.7-7.1 lesions/kilobase pair/microM drug) in several prominent AT islands, compared with markedly lower lesion levels in several motif-poor loci and in bulk cellular DNA (approximately 0.8-1.3 and approximately 0.9 lesions/kilobase pair/microM drug, respectively). The identified AT islands exhibit sequence attributes of matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. The computed "MAR potential" and propensity for supercoiling-induced duplex destabilization (both predictive of strong MARs) correlate with the total number of bizelesin binding sites. Hence, MAR-like AT-rich non-coding domains can be regarded as a novel class of critical targets for anticancer drugs.

Development of ribozymes that target stathmin, a major regulator of the mitotic spindle.

Stathmin is a major cytosolic phosphoprotein that plays an important role in the control of cellular proliferation by regulating the dynamics of the microtubules that make up the mitotic spindle. Because stathmin is expressed at high levels in all human cancers, it is an attractive molecular target for anticancer interventions. We had shown previously that antisense stathmin inhibition results in marked abrogation of the transformed phenotype of leukemic cells in vitro and in vivo. Unlike the antisense approach, ribozymes can catalytically cleave several molecules of target RNA. This may provide a more efficient strategy for downregulating genes, such as stathmin, that are expressed at very high levels in cancer cells. We designed several antistathmin hammerhead ribozymes and tested their cleavage activity against short synthetic stathmin RNA substrates. In vitro cleavage studies demonstrated site-specific cleavage of stathmin RNA that was dependent on ribozyme concentration and duration of exposure to ribozyme. The most active antistathmin ribozyme was capable of cleaving >90% stathmin RNA in a catalytic manner, cleaving multiple substrate molecules per ribozyme molecule. We also demonstrated that the designed antistathmin ribozymes are capable of selectively cleaving native stathmin RNA in a mixture of total RNA isolated from leukemic cells. These antistathmin ribozymes may provide a novel and effective form of gene therapy that may be applicable to a wide variety of human cancers.

Stress-induced DNA duplex destabilization in transcriptional initiation.

Stress-induced destabilization of the DNA double helix (SIDD) is involved in several mechanisms by which transcription is regulated. This paper describes a computational method for predicting the locations and extents of destabilization as functions of DNA sequence and imposed superhelical stress. This method is used to investigate several transcriptional regulatory events. These include IHF-mediated activation of gene expression in E. coli, the bimodal control of the initiation of transcription from the human c-myc gene, and the determination of the minimal requirements for transcriptional activity in yeast. Collaborations with experimental groups have established the central role of SIDD in each of these processes.

Neuroprotective role of monocarboxylate transport during glucose deprivation in slice cultures of rat hippocampus.

The effects of energy substrate removal and metabolic pathway block have been examined on neuronal and glial survival in organotypic slice cultures of rat hippocampus. Slice cultures resisted 24 h of exogenous energy substrate deprivation. Application of 0.5 mM alpha-cyano-4-hydroxycinnamate (4-CIN) for 24 h resulted in specific damage to neuronal cell layers, which could be reversed by co-application of 5 mM lactate. Addition of 10 mM 2-deoxyglucose in the absence of exogenous energy supply produced widespread cell death throughout the slice. This was partly reversed by co-application of 5 mM lactate. These effects of metabolic blockade on cell survival were qualitatively similar to the effects on population spikes recorded in the CA1 cell layer following 60 min application of these agents. The data suggest that monocarboxylate trafficking from glia to neurons is an essential route for supply of energy substrates to neurons particularly when exogenous energy supply is restricted.

An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled.

Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

Characterisation of SB-221420-A - a neuronal Ca(2+) and Na(+) channel antagonist in experimental models of stroke.

For progression to clinical trials in stroke, putative neuroprotective compounds should show robust efficacy post-ischaemia in several experimental models of stroke. This paper describes the characterisation of (+)(1S, 2R)-cis-1-[4-(1-methyl-1-phenylethyl)phenoxy]-2-methylamino indane hydrochloride (SB-221420-A), a Ca(2+) and Na(+) channel antagonist. SB-221420-A inhibited (IC(50)=2.2 microM) N-type voltage-operated Ca(2+) channel currents in cultured superior cervical ganglion neurons, which were pretreated with 10 microM nimodipine to block L-type voltage-operated Ca(2+) channel currents. In dorsal root ganglion neurons pretreated with 1 microM omega-conotoxin GVIA to block N-type voltage-operated Ca(2+) channel currents, SB-221420-A inhibited the residual Ca(2+) current with an IC(50) of 7 microM. SB-221420-A also inhibited Na(+) currents in dorsal root ganglion neurons with an IC(50) of 8 microM. In rats, the pharmacokinetic profile of SB-221420-A shows that it has a half-life of 6.4 h, a high volume of distribution, is highly brain penetrating, and has no persistent metabolites. Following bilateral carotid artery occlusion in gerbils, SB-221420-A significantly reduced the level of ischaemia-induced hyperlocomotor activity and the extent of hippocampal CA1 cell loss compared to the ischaemic vehicle-treated group. SB-221420-A was also effective in focal models of ischaemia. In the mouse permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously, post-ischaemia significantly (P<0.05) reduced lesion volume compared to the ischaemic vehicle-treated group. In the normotensive rat permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously over 1 h, beginning 30 min postmiddle cerebral artery occlusion, significantly (P<0.05) reduced lesion volume from 291+/-16 to 153+/-30 mm(3), compared to ischaemic vehicle-treated controls when measured 24 h postmiddle cerebral artery occlusion. Efficacy was maintained when the same total dose of SB-221420-A was infused over a 6-h period, beginning 30 min postmiddle cerebral artery occlusion. SB-221420-A also significantly (P<0.05) reduced lesion volume following transient middle cerebral artery occlusion in normotensive rats and permanent middle cerebral artery occlusion in spontaneously hypertensive rats (SHR). Investigation of the side effect profile using the Irwin screen in mice revealed that, at neuroprotective doses, there were no overt behavioural or cardiovascular changes. These data demonstrate that robust neuroprotection can be seen post-ischaemia with SB-221420-A in both global and focal ischaemia with no adverse effects at neuroprotective doses, and indicate the potential utility of a mixed cation blocker to improve outcome in cerebral ischaemia.