In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders. Convulsive disorders affect a significant number of people, with a rate of prevalence of 1% in the general population, despite the availability of more than 20 FDA approved anti-convulsive drugs The natural monoepoxide metabolites of major polyunsaturated fatty acids are bioactive molecules involved in the regulation of neuronal excitability, in particular during pathological processes in vivo stabilize EpFAs and allow the quantification of their effects on physiologic processes, since in the presence of sEH activity the half life of EpFAs are in the order of seconds The intracellular concentration of the free EpFAs are tightly regulated by the enzyme soluble epoxide hydrolase (sEH) which converts EpFAs to their corresponding diols, molecules that are inactive or in some cases seem to oppose the activities of EpFAs Possible effects of EpFAs on GABA-ergic transmission may be through neurosteroids All studies were conducted in line with federal regulations and were approved by the Institutional Animal Care and Use Committee at University of California, Davis.meta-chloroperbenzoic acid (mCPBA) and purified by reverse phase HPLC as described previously Synthesis of sEH inhibitors TUPS (1-(1-methylsulfonylpiperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea) and TPPU (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea) were previously reported The 11(12)-EET*d8 and PGD2*d4 were purchased from Cayman Chemical . Omni-Solv™ acetonitrile and methanol purchased from EM Science  and were used for all LC-MS/MS analyses. Other chemicals and supplies were obtained from Fisher Scientific .EPHX2) which is backcrossed to C57BL/6 background was generated as described earlier Age matched male NIH Swiss mice weighing 25–30 g were obtained from The National Cancer Institute Animal Production Program at Frederick National Laboratory . A colony of sEH−/− mice with targeted deletion of sEH gene  and pentylenetetrazol (PTZ) tests, 10 mg/kg PIC and 80 mg/kg of PTZ were administered by subcutaneous route, time to onset of first clonic seizure, time to tonic hind limb extension and lethality were monitored for a duration of 45 min for PIC and 30 min for PTZ. For the subcutaneous 4-aminopyridine test 13 mg/kg 4-AP was given subcutaneously, time to onset to tonic hind limb extension and lethality were monitored for 30 min. For the PTZ threshold test a 10 mg/mL solution of PTZ was infused via the tail vein of lightly restrained mice at a rate of 0.5 mL/min and the threshold to the onset of tonic extension was quantified. The threshold value (milligrams per kilogram) was determined according to the following formula: (infusion duration [seconds]×infusion rate [milliliters per minute]×convulsant drug concentration [milligrams per milliliter]×1000)/(60 s×weight of mouse [grams]). For the intraperitoneal (i.p.) PTZ seizure test, PTZ was administered by i.p route and parameters as outlined above were quantified. Two structurally different inhibitors of sEH were completely dissolved completely using PEG400 to give clear solutions as described earlier. Inhibitors or vehicle were administered subcutaneously 1 h prior to pro-convulsants at a range of doses. For the subcutaneous PTZ seizure test, diazepam injected in its own vehicle containing saline, 40% propylene glycol, 10% ethanol and the neurosteroid allopregnanalone  dissolved in PEG400 served as positive control. For the antagonism assay, finasteride was dissolved in PEG400 and administered 1 h prior to sEH inhibitor at a single dose of 10 mg/kg.To investigate the direct roles of epoxy fatty acids the EETs, EpETEs and EpDPEs, diazepam (0.5 µg each), and sEH inhibitor TUPS (0.1 and 1 µg) were administered to mouse brain. Mice were anesthetized for 2 min in an induction chamber using isoflurane (3% with 2 LPM oxygen). Compounds were dissolved in vehicle (PEG400) and administered in a final volume of 1 µL into the cerebroventricular (i.c.v) space using a 22- gauge needle connected to a 10 µL hamilton syringe with a PE50 polyethylene tubing. The needle was removed 30 sec after injection was completed so as to prevent outflow and allow dispersion of the injected material. Mice fully recovered from anesthesia within 2 min indicated by responses to light touch and the presence of natural exploratory behavior. Thirty minutes following these treatments, i.p. PTZ (80 mg/kg) test was performed and seizure related parameters were quantified as described above.Testing was carried out as previously described Animals were subjected to a 0.2-s, 60-Hz electrical stimulus through corneal electrodes. The electroshock unit was adjusted to deliver a constant current of 50 mA. Animals failing to show tonic hind limb extension were scored as protected Inhibitor levels were quantified from blood and brain samples of treated animals using previously described methods g rpm for 10 min at 4°C. The supernatants were collected and pellets were re-extracted with 400 µL of ice-cold methanol with 0.1% of acetic acid and 0.1% of BHT and re-centrifuged. The supernatant of two extractions were combined and loaded onto preconditioned solid phase extraction cartridges , partially purified and reconstituted in 50 µl methanol as described previously. A 5 µL aliquot was directly analyzed by LC-ESI-MS/MS as described previously. The limit of quantification (LOQ) of the method was defined as the quantity of sample required to produce a signal-to-noise ratio (S/N) of at least 10. Typically calibration standards had LOQs between 0.07 and 32 pg for the deuteriated analytes as reported earlier.Eicosanoid analysis was performed on brain samples taken immediately following tonic seizures. Brains were removed; one hemisphere from each brain was rapidly frozen on dry ice, weighed and re-suspended in a solution containing 10 µL of anti-oxidants (0.2 mg/mL of BHT and EDTA), 10 µL of surrogate solution and 400 µL of ice-cold methanol with 0.1% of acetic acid and 0.1% of BHT. These samples were incubated at −80°C overnight for extraction. The next day, an internal standard solution (10 µL) containing deuterated standards was added and samples were homogenized using an ultrasonic-homogenizer at 30 Hz for 10 min and centrifuged at 10,000×Data were analyzed by non-parametric and parametric One way-ANOVA followed by appropriate post hoc tests for between group comparisons selected by the software suite SigmaPlot analysis package . Results are presented as mean ± standard error of mean.A complex are overlapping but not identical Several prior observations led to the hypothesis that at least part of the analgesic effects of EpFAs and inhibitors of sEH may be through a GABA mediated mechanism andThese observations prompted us to investigate if a comparable phenotype could be recapitulated by using potent small molecule inhibitors of sEH. Dose dependent effects of two structurally different but highly potent inhibitors TUPS and TPPU were studied and more detailed characterization was carried out using TUPS. In the subcutaneous PTZ assay, both inhibitors significantly delayed the onset of tonic hind limb extension and reduced lethality when administered 1 h prior to PTZ by intraperitoneal (i.p.) route andTable 1Next, PTZ threshold test was performed to ask if stabilization of EpFAs by pretreatment with sEHI would change acute seizure threshold. The threshold test is highly consistent and eliminates the potential confounding effects of the pharmacokinetics of PTZ, thus is used for comparison of the anticonvulsant potency of therapeutic candidates. Pre-treatment with two structurally different sEHI resulted in significant increase in threshold for tonic hind limb extension for both compounds supporting the earlier observations Fig. 3. Earlier data on anti-nociceptive effects of sEH inhibitors suggested that at least part of the analgesic activity could be mediated by a neurosteroid mediated mechanism in vitro IC50 value of TUPS  throughout the 8 h observation period . Although the levels of the bioactive regioisomer, 14,15-EET, did not display a significant change in response to the inhibitor, it was significantly diminished by seizure activity and recovered in PTZ exposed and sEHI treated seized mice . The different responses of various EpFAs to sEHI and seizures suggest distinct regulatory mechanisms for individual lipid metabolites.Next we asked if inhibition of sEH in the brain, the site of action for anticonvulsant drugs, modulated the levels of bioactive lipids. Brains were excised immediately following tonic seizures or 30 min following i.p. saline under deep anesthesia for control groups, frozen on dry ice extracted on the same day and stored until analysis. The ratio of epoxy- to dihydroxy- eicosanoids (epoxide to diol ratio) is used as a marker of sEH inhibition Fig. S2). Although the role of each prostanoid in seizures is not clear, in general there is a consensus about pro-convulsant or seizure enhancing role of PGE2, while PGD2 is thought to have the opposite effect. Regardless, inhibition of sEH by genetic knockout or by sEHI resulted in a highly consistent profile of increased brain levels of PGE2 and PGD2 which was not reduced in sEH−/− or sEHI treated animals . These results demonstrate that the effect of sEH inhibition is independent of prostanoid levels. These findings are contrary to the extensive effects of sEHI on prostanoid levels in models of peripheral inflammation Consistent with previous observations there was a general increase in the brain levels of major prostanoids in wild type and sEH−/− mice following tonic seizures induced by PTZ and 4-AP . These data overall demonstrate that the majority of the biological activity of sEH inhibitors is mediated by elevation of EETs in the brain.If inhibition of sEH is anticonvulsant by elevating the brain levels of EpFAs then direct administration of EpFAs and sEHI into the brain should also delay the onset of seizures. Therefore we tested anticonvulsant effects of direct administration of EpFAs and sEHI into the mouse brain. Mice were lightly anesthetized, compounds delivered by i.c.v. route in 1 uL volume, and PTZ (80 mg/kg) administered i.p. 30 min after i.c.v. injection. A low dose of diazepam was used as a positive control. Consistent with the changes in the brain levels of EpFAs, only the regioisomeric mixture of EET methyl esters (epoxy-ARA metabolites) but not EpDPE or EpETE methyl esters delayed the onset of clonic and tonic seizures Fig. 6. de novo and rapidly degrade these molecules via sEH activity. Region specific existence of various EpFAs and sEH in the CNS has also been reported The levels of the bioactive EpFAs are controlled by several distinct mechanisms in vivo, strongly indicating that the mechanism of action of sEHI is through stabilization and prolonging the activity of the EpFAs A number of structurally different but potent small molecule inhibitors of sEH have been demonstrated to stabilize the EpFAs A antagonists PIC and PTZ are impeded by inhibition or deletion of sEH is of significance. Equally important, these effects seem to be selective towards GABAA receptors. Given that 4-AP induced seizures were consistently unaffected by inhibition of sEH, the observed effects should not be driven by the potential ability of EETs to modulate BKCa mediated hyperpolarization in neurons A-mediated mechanism of action in the antihyperalgesic effects of EpFAs and sEHI are in agreement with this study The finding that convulsions produced by GABAAlthough it is unclear if EpFAs enhance neurosteroid synthesis and thus enhance GABA-ergic inhibition, more evidence towards the presence of an interaction between EpFAs and the GABA system is presented here. More to the point, finasteride, a prototypical inhibitor of 5α-reductase partially blocked the effects of sEHI, while allopregnanolone enhanced these effects. Importantly, these results are similar to those obtained in pain assays suggesting a common mechanism of sEHI mediated suppression of neuronal excitability.Fig. S2). These increases in prostanoids validate our LC-MS/MS based approach. However, surprisingly the changes were asymmetric among the products of the three branches of the ARA cascade, with increases only in prostanoids and variable levels in cytochrome P450 products. This indicates a degree of dynamic regulation in the synthesis of bioactive lipids in the brain. An advantage of our approach is that all metabolites are analyzed together, thus a more comprehensive view of the biological processes is obtained. A limitation of this study however is that one hemisphere of the brain was analyzed, thus we are unable to determine the loci of the changes. Levels of EETs vary by brain region, therefore our results likely underestimate local changes in response to sEH inhibition Given the effects mediated by sEH inhibition in assays of seizures one would expect to see corresponding changes in the levels of EpFAs in the brain, the site of convulsions. Our analysis of mouse brain demonstrates expected changes in the brain levels of EpFAs in response to sEH inhibition. Nevertheless, it was surprising to observe highly selective, regioisomer specific changes in the levels of EpFAs. Although metabolites of ARA, including prostanoids were generally increased the bioactive regioisomer 14,15-EET was reduced along with all DHA derived EpFAs, yet EPA derived EpFAs were elevated after seizures. The basis of these changes were not addressed in this study, though these observations justify further work to enhance our understanding of mechanisms regulating free fatty acids and their metabolites in the brain. Ictal activity leads to the release of ARA from membrane stores 2 and PGD2. This observation adds to the argument that fundamentally different mechanisms underlie anticonvulsant and anti-inflammatory activities of sEH inhibition.In contrast to earlier findings in acute inflammatory models in which inhibition of sEH leads to suppression of transcriptional upregulation of COX-2 and consistently reduces the levels of major prostanoids, here genetic or chemical inhibition of sEH had no effect on the levels of brain PGELastly, these results encouraged us to test EpFAs directly by i.c.v. administration. The regioisomeric mixture of cell permeable EET-methyl esters were exclusively active in delaying onset of tonic seizures while the epoxy-DHA and -EPA metabolites were largely devoid of effects in the brain. The parallel results obtained with EETs and sEHI following i.c.v. administration are consistent with that of systemic sEHI and experiments using sEH−/− mice. The EETs were as potent as the low dose of diazepam in this case, though their intrinsic activity is likely to be lower than that of an effective dose of diazepam. Data from this study suggests that more effort into understanding the effects of EETs in the brain is warranted and will likely yield valuable information on how EETs and other EpFAs modulate physiology of the brain, particularly under excitatory conditions. In particular presence of sufficient amounts of EpFAs may prevent synchronized discharges as suggested by the observation that in sEH−/− and i.c.v. EET treated mice, tonic extension reflex was not consistently observed. An interesting future direction, and a limitation of this study, is the quantification of EpFAs following clonic seizures to determine if the reduction in the levels of EpFAs is because of clonic or tonic seizures.EpFAs and sEH inhibitors display numerous positive effects in multiple disease models. Here we demonstrate yet another mechanism of action for EpFAs that has not been shown previously. Although molecular target(s) for EpFAs are not yet identified, the structural diversity of EpFAs that are stabilized by preventing their degradation may, based on concentration and context, lead to modulation of different receptors. In this case, a clear effect on GABA mediated excitotoxicity is linked with only one group of EpFAs, the EETs. This type of selectivity also argues that sEH inhibition leads to context based pleiotropic effects because one modulates a diverse set of EpFAs with sEHI.Even though rodent models of convulsions are considered to be clinically relevant, an estimated one third of patients with epilepsy are thought to suffer from intractable or pharmaco-resistant epilepsy Figure S1Brain levels of major EpFAs and their corresponding dihydroxy- metabolites change after PTZ induced seizure. Following tonic hind limb extension the brain levels of sum of EpFAs and their corresponding dihydroxy- metabolites from ARA , DHA  and EPA  showed distinct patterns indicative of sEH dependent and independent mechanisms that selectively regulates their levels. (A) The sum of EET regioisomers remained unchanged in response to seizure but were elevated by inhibition of sEH with a concomitant decrease in the sum of corresponding dihydroxy- metabolites. (B) The sum of EpDPE regioisomers were decreased following seizures but were unchanged in response to inhibition of sEH. (C) The sum of EpETE regioisomers and their corresponding dihydroxy metabolites increased in response to seizure. While inhibition of sEH did not further elevate the EpETEs TUPS reduced the levels of the corresponding dihydroxy metabolites.(TIF)Click here for additional data file.Figure S2Brain levels of major prostanoids increase following tonic hind limb extension induced by PTZ and 4-AP. (A) In wild type and sEH−/− mice brain levels of major prostanoids display a highly similar profile in which PGE2, PGD2 and PGF2α and PGJ2 are elevated to the same degree. This data suggests brain sEH had no role in modulating the levels of prostanoids in the brain after seizures. (B) Parallel results are obtained with small molecule inhibitor of sEH. (C) Seizure induced by 4-AP also led to an increase in prostanoids although these increases were smaller in magnitude than PTZ induced changes.(TIF)Click here for additional data file.Figure S3Direct administration of EETs and sEHI into the brain delay onset of PTZ induced seizures. Bar graph of percent change in onset of clonic (black bars) and tonic (gray bars) seizures following intracerebroventricular EETs and TUPS (n = 6–14mice/group) 10 min post administration (see (TIF)Click here for additional data file.Table S1Structure and properties of sEHI used in the study.(TIF)Click here for additional data file.


Dopamine is still frequently used as a first line vasopressor agent in hypotensive patients, when physicians are afraid of noradrenaline and believe that dopamine, with its β and α, inotrope and vasopressor effects, may be helpful. Evidence exists that it does not offer protection from renal failure, even if at low doses  it may exert its effects on D1 and D2 receptors resulting in natriuresis and renal vasodilation, augmentation in renal blood flow, and diuresis.The effects of dopamine on gastrointestinal system and splanchnic perfusion in critical care patients are even more controversial, since they seem to be at least partially dependent on the initial fractional splanchnic blood flow. Dopamine may exert deleterious effects on respiratory function, by impairing the ventilatory drive response to hypoxemia and hypercapnia and reducing arterial oxygen saturation through a regional ventilation/perfusion mismatching. Dopamine seems to affect the cellular mediated mechanism of the immune function directly by its action on receptors located on immune system cells and indirectly altering the hormonal response regulating immune response. In this paper, the use of low dose dopamine is discussed in the intensive care perspective. I.Dopamine (DA) remains an essential drug in Intensive Care Units (ICU), where it is still used as a first line vasopressor agent in hypotensive patients, refractory to fluid resuscitation, because of the feared ischemic side-effects of norepinephrine on end-organ perfusion.Nevertheless, the results of the SOAP study showed that dopamine administration in shock patients, compared to patients who did not receive it, was associated with 20 % increase in ICU and hospital mortality rates . AlternaPharmacology of dopamine is interesting: its peculiar spectrum of action resides in the dose dependent interaction to different catecholamine receptors.At low doses (0.3–5 μg/Kg/min) DA exerts its effects on D1 and D2 receptors resulting in, as the kidney is concerned, natriuresis and renal vasodilation, augmentation in renal blood flow, and diuresis.Dose dependent interactions on different receptors are not a clear cut-off value but represent the prevalence of activation of a group of receptors over another with a wide range of inter-individual variability. Thus, even a low dosage of dopamine may exert a systemic vasocostrictory action without relevant improvement in renal function . A low dThe optimal selection of dopamine dosages is far less clear in critical care settings where an altered receptor function and responsiveness make the individual response unpredictable.II.The era of dopamine, particularly “low dose dopamine” (LDD), began in 60’s when Goldberg described its effects on four patients affected by end stage congestive heart failure . Drug adThe same investigators showed that dopamine administration was able to increase plasmatic flow in the kidney, glomerular filtration, and sodium excretion in healthy human subjects . In thisIn 1965, the same authors investigated the renal effects of dopamine in anaesthetised dogs and concluded that dopamine might exert its action on particular receptors located in the kidneys . Twenty The dopamine suppressor dose was at that time defined as the dose at which dopaminergic and possibly adrenergic stimulation prevailed over adrenergic stimulation. This threshold corresponded to the infusion rate: < 5g/kg/min .Dopamine exerts its effects on the kidneys in dose dependent fashion.At low doses, such as 0.3–5 μg/Kg/min, dopamine acts on D1 vascular receptors, which in turn increases renal blood flow. It appears that dopamine may additionally interact with D2 receptors located on presynaptic nerve endings, inhibiting the release of norepinephrine . At highDopamine is able to induce diuresis and natriuresis by acting on both D1 and D2 receptors located on the proximal tubule, which is the thick ascending loop of the Henle and cortical collecting tubule.+/K+-adenosine triphosphatase activity. In fact, it appears that the primary effect on renal epithelial cells is the removal of the plasma membrane of active Na+/K+ ATPase units. The net effect is the reduced capability of the tubular cells to Na+ transport [Those effects are achieved by the inhibition of Naransport .2, (PGE2), which counterbalances the effects of antidiuretic hormones, augmenting the clearance of free water [Moreover, the stimulation of D2 receptors located on the collecting tubules of the inner medulla stimulates production of prostaglandin Eee water , 15.The renal vasodilatory effects are associated with dose-dependent augmentation in renal blood flow and diuresis.2 and shunting blood away from the outer medulla [LDD induces a redistribution of intraparenchymal renal blood flow towards the cortical region, counteracting the effect of PGE medulla .This can be harmful for two reasons. First, renal medulla has a limited blood supply. Second, it may promote a relative ischemia in a region that is high metabolically active and already works with a lower tension of oxygen.In fact, although the kidneys receive nearly 20 per cent of cardiac output, the greatest part of the blood flow supplies the outer parenchymal layers .For years LDD was a widely accepted therapeutic option to limit or prevent acute renal failure in critical care patients, especially those affected by sepsis. Even if largely studied, sepsis is still the greatest danger for these patients’ life, with many obscure sides on its presentation, causes and prevention possibilities , 19. SevIn some studies, LDD administration increased urine output; however, in others, no effect was found , 20–23.One study showed a potential negative effect of LDD dopamine administration on tubular function caused by the augmented urinary excretion of retinol binding protein in patients who had undergone coronary bypass surgery . AnotherIn fact, there is convincing evidence from literature that LDD not only is unable to prevent, reverse, or limit the progression of acute renal failure (ARF), but its use, regardless of a clear assessment of the volemic status of the patients, may increase the risk of ARF.Moreover, a large prospective randomized study by the Australian and New Zealand Intensive Care Society Group showed that LDD not only was unable to prevent or reverse acute renal failure, but it failed to improve outcome variables.In fact, there were no differences in terms of mortality, need of renal replacement therapy, renal recovery, and peak serum creatinine among the patients.These findings confirmed the results of the retrospective analysis of the North American Septic Shock Trial (NORASEPT), where no reduction of the incidence of acute renal failure, the 28-day mortality, nor the requirement of haemodialysis were observed in septic patients who developed oliguria .In two recent meta-analyses about the impacts of LDD on ARF, the first by Kellum and Decker, dopamine did not prevent mortality, the onset of acute renal failure, or the need for haemodialysis . The secIt has been argued by some authors that adding LDD in patients requiring norepinephrine may limit its adverse effects on renal circulation and function.Clear beneficial evidence on renal function of this therapeutic regime is lacking, as shown by studies carried out on experimental animal models and in patients with septic shock who require catecholamine administration.It seems clear that LDD mediated increases in urinary output in septic shock patients treated with norepinephrine are probably mediated by the augmentation of cardiac output.Recent evidence has shown that norepinephrine administration can effectively restore an adequate hemodynamic status in adequately fluid resuscitated patients .The use of norepinephrine has been shown to have a protective effect on renal blood flow and to increase diuresis in animal and human septic shock conditions.A low dosage dopamine appears to be able to increase in urinary output in critically ill patients, but it doesn’t play any protective role against acute renal failure and does not improve the course of an established acute renal failure.When administered to critical patients, it may increase the risk of acute renal failure.It could be interesting, but far from the topic of this review, to consider the use of new molecules in combination with dopamine, as vaptans i.e .III.Gut has been considered as the “motor” of systemic inflammatory response syndrome (SIRS) . In factIn experimental models, dopamine increased both splanchnic ad hepatic blood flow. In a study on dogs, dopamine reduced intestinal blood flow, and in a porcine model, it seemed to hasten gut ischemia.Those results seem to be due to the ability of DA to reduce blood flow to the mucosa by redistributing it within the gut.In another animal study DA improved mucosal blood flow and oxygenation .Data regarding human studies shared the same deal of equivocal conclusion.In fact, some investigations showed that LDD can increase splanchnic blood flow in septic cardiac surgical patients, whereas others did not draw the same results –35.LDD seemed to decrease splanchnic oxygen consumption in septic patients in spite of an increase in splanchnic blood flow, and once again this effect was not confirmed in cardiac surgical patients.LDD increased oxygen transport in septic patients but led to a diminished gastric mucosal flow and did not affect pHi, a common and widely accepted marker of gut mucosal perfusion.The effect of DA administration seems to be at least partially dependent on the initial fractional splanchnic blood.et al. found no differences in PCO2 gap, splanchnic blood flow in their study, which was carried out on 20 septic patients. Moreover, dopamine administration showed a lower mixed venous-hepatic venous saturation gradient.Recently, De Backer DA 2 receptors are present in human enteric nervous endings, and dopamine administration may actually affect gastrointestinal motility.These effects have been confirmed both in healthy subjects who had undergone short-term DA administration, and in critically ill patients, in doses ranging from 2.5 to 5 μg/kg/min. Moreover, in another paper, LDD impaired gastroduodenal emptying in mechanically ventilated patient during fasting and nasogastric enteral feeding . MoreoveIV.Intensive care patients are very often mechanically ventilated being respiratory failure the cause of their recovery in ICU or a complication of their illness –40. As pProblems can arise during the weaning process from ventilatory support, when the physiological response to both hypoxia and hypercapnia might have been blunted by DA administration. LDD may favour weaning from the ventilator, but this comes at the expense of an actual risk of hastening respiratory failure.V.Low doses of dopamine result in plasmatic levels up to 100 times higher than those generated by endogenous secretions, which may cause partial hypopituitarism in adults, infants, and children. In a work by Van den Berghe  on 12 poLDD infusion may trigger or exacerbate the euthyroid sick syndrome in critical illnesses.DA administration affected the secretion of the growth hormone (GH) in critically ill patients as pointed out by the same authors. GH pulsatile secretion is impaired in critically ill patients and it resulted further by being blunted by DA administration. The authors concluded that the suppression of GH secretion might enhance the catabolic process of critical illnesses.LDD dopamine has been show to suppress dehydroepiandrosterone (DHEAS) and prolactine levels in 20 critically ill patients .Cortisol levels were not affected. The levels of luteinizing hormone (LH) and testosterone were affected by dopamine administration in 15 critically ill men.LH rose after three hours from dopamine withdrawal, while testosterone levels failed to rebound.Dopamine receptors have been discovered on thymocytes, and dopamine is able to interact with lymphocytes. Dopaminergic agonist and dopamine suppress T-lymphocytes function and suppress T-cell functions in mice. In humans, specifically critically ill patients receiving dopamine, the drug was able to reduce the T-cells’ responsivenessProlactine, whose levels have been shown to decrease under dopamine infusion, has immunoregolatory functions; B and T types have indeed prolactine receptors.The reduction of DHEAS has been advocated as a further cause of immune cellular response, because of lymphocyte T-helper and type 1-T lymphocyte induced dysfunction.Dopamine seems to affect the cellular mediated mechanism of the immune function directly by its action on receptors located on immune system cells and indirectly altering the hormonal response regulating immune response.VI.Dopamine “owes” its popularity to the work of Goldberg. Over the years, renal and splanchnic protective effects have been challenged and not confirmed at all. There is no equivocal conclusion about its effects on gut, but DA has been proven to cause major disturbances in anterior pituitary function and the immune system and may further impair muscular blood supply.We can conclude that low dose dopamine for renal protection is no longer justified and should be definitely abandoned. Its use as a first choice vasopressor should be questioned in view of its potentially deleterious side effects and the increased rate of mortality associated with its administration in septic patients. However, DA is still used by medical personnel; many doctors are familiar with the medication and feel comfortable for its application, therefore, it becomes part of their routine treatment: in clinical situations, very often, the choice of catecholamine is based on personal and cultural preferences, not evidence based.


Caenorhabditis elegans, the release of dopamine during behavioral plasticity is in part modulated through an acid-sensing ion channel expressed in its eight dopaminergic neurons. A D2-like C. elegans dopamine receptor DOP-2 co-expresses along with a Gαi subunit (GPA-14) in the anterior deirid (ADE) pair of dopaminergic neurons.Precise levels of specific neurotransmitters are required for appropriate neuronal functioning. The neurotransmitter dopamine is implicated in modulating behaviors, such as cognition, reward and memory. In the nematode in vitro physical interaction between DOP-2 and GPA-14, we have behaviorally characterized worms carrying deletion mutations in gpa-14 and/or dop-2. We found both mutants to display behavioral abnormalities in habituation as well as associative learning, and exogenous supply of dopamine was able to revert the observed behavioral deficits. The behavioral phenotypes of dop-2 and gpa-14 loss-of-function mutants were found to be remarkably similar, and we did not observe any cumulative defects in their double mutants.In follow-up experiments to our recently reported in vitro results where we had shown that the DOP-2 dopamine receptor and the GPA-14 Gαi subunit physically interact with each other. Results from behavioral experiments presented here together with our previous in-vitro work suggests that the DOP-2 functions as a dopamine auto-receptor to modulate two types of learning, anterior touch habituation and chemosensory associative conditioning, through a G-protein complex that comprises GPA-14 as its Gα subunit.Our results provide genetic and phenotypic support to our earlier  Faster mechanosensitive habituation has been reported for mutants in dop-1, which codes for a D1-like receptor  may give an impression of contradictory phenotypes. While diminished associative learning clearly correlates with poorer neuronal summation, faster habituation does not necessarily draw a parallel with an antagonistic explanation. On the other hand faster habituation may correlate with ignoring a cue earlier than optimum assuming that WT habituation represents an optimal rate. In summary, examining the behavioral profile of dop-2(vs105) and gpa-14(pk347) deletion mutants has revealed that both show similar aberrations in plasticity, and our results with the dop-2;gpa-14 double mutant indicate that GPA-14 and DOP-2 work together in the same pathway. In order to rule out the contribution of background mutations, follow-up experiments using RNAi knockdowns or transgenic rescue of the mutants can provide required validation of the results presented here.Absence of any additional or cumulative abnormalities in the i subunit [These results are significant in that neuroimaging studies in humans indicate that deficient feedback monitoring in the cortex is associated with learning deficits due to D2 receptor polymorphisms ,26. The  subunit ,21. Alte subunit . Furthergpa-14; dop-2: Gene for D2 like dopamine receptor; DOP-2: dop-2 protein product; ASIC: Acid sensing ion channel; asic-1: Gene for a subunit of C. elegans ASIC.gpa-14: Gene for G-protein alpha subunit-14; GPA-14: Protein product coded by The authors declare that they have no competing interests.Conceived project: NT, SH. Designed experiments: MM, RF, PP, SH. Performed experiments: MM, RF, RM. Wrote manuscript: NT, SH. All authors have read and approve the final manuscript.Basal slowing rates for dop-2(vs105) and gpa-14(pk347) mutants do not differ from wild type animals. Body bends of 3 day old individual worms were counted for 20 seconds. Basal slowing assay with food is represented by the white bars while basal slowing assay without food is represented by the grey bars. Each bar indicates the average body bends/20 seconds in three experiments. Error bars indicate SEM .Click here for filegpa-14(pk347) deletion mutants displayed associative learning deficits when paired with either soluble or volatile chemicals, isoamyl alcohol or sodium chloride, respectively. n = 90 in three experiments; *: P < 0.001; ns: non-significant; unpaired t-test.Click here for fileA model for the molecular interactions modulating neurotransmitter levels at a C. elegans dopaminergic synapse {modified from [}. Release of dopamine can activate auto-receptor function of DOP-2 to initiate signal transduction through GPA-14 ([+ ions from the acidified vesicles activates acid sensing cation channels (ASIC). Stimulation of DOP-2 and activation of ASIC call allow two molecular loops in the presynaptic neuron so as to modulate levels of dopamine in the synaptic cleft. These two pathways are likely to crosstalk through relay molecule/s that remain unknown as yet.ied from }. Releas GPA-14 (, and thiClick here for file


This work investigated the association of acute ethanol and aminophylline administration on behavioral models of depression and prefrontal monoamine levels (i.e. norepinephrine and dopamine) in mice. The animals received a single dose of ethanol (2 g/kg) or aminophylline (5 or 10 mg/kg) alone or in association. Thirty minutes after the last drug administration, the animals were assessed in behavioral models by the forced swimming and tail suspension tests. After these tests, the animals were sacrificed and the prefrontal cortices dissected to measure monoamine content. Results showed that ethanol presented depression-like activity in the forced swimming and tail suspension tests. These effects were reversed by the association with aminophylline in all tests. Norepinephrine and dopamine levels decreased, while an increase in the dopamine metabolite, (4-hydroxy-3-methoxyphenyl)acetic acid (DOPAC), after ethanol administration was observed. On the contrary, the association of ethanol and aminophylline increased the norepinephrine and dopamine content, while it decreased DOPAC when compared to the ethanol group, confirming the alterations observed in the behavioral tests. These data reinforce the involvement of the adenosinergic system on ethanol effects, highlighting the importance of the norepinephrine and dopamine pathways in the prefrontal cortex to the effects of ethanol. Ethanol differentially interferes with the transmission processes in the central nervous system, affecting neurotransmitters , 2 and lAmong the wide range of pathways in the central nervous system that are modified by ethanol, it is important to highlight those that underlie ethanol’s diverse effects, like the ones related to the release of gamma-amynobutiric acid (GABA), glutamate, dopamine, and norepinephrine , 12. Mor1 receptor, which is a presynaptic inhibitor of the release of neurotransmitters such as dopamine, GABA, glutamate, acetylcholine, and norepinephrine  .The levels of NE, DA, and DOPAC in the prefrontal cortex are presented in Analyzing the monoamines, metabolites, and ethanol (677.6 ± 59.1) increased the DOPAC levels as compared to the control (C: 362.8 ± 67.2) and the association groups (E/A5 or E/A10). This drug was also effective in the reversal of the metabolite alterations caused by ethanol, reducing the DOPAC concentration when compared to the ethanol group  .We investigated the effects of the association of acute ethanol and aminophylline administration in behavioral models for the assessment of depression-like activity, i.e. forced swimming and tail suspension tests, which are predictive models of clinical antidepressant activity. Because regulations in monoamine levels are related to depression-like activity, we also assessed monoamine levels in the prefrontal cortex.In the present work, the administration of ethanol in mice induced a depression-like behavior, as indicated by a significant increase in the immobility time in the forced swimming and tail suspension tests. The comorbidity of substance use disorders (for example ethanol) and depression is highly prevalent in the general population , 33, 34.2A receptor knockout mice displayed reduced immobility in functional assays in vivo, such as in the tail suspension and forced swim tests [Aminophylline is a non-selective adenosine receptor antagonist , 36. Theim tests .In addition, an increasing body of evidence points to a direct role of adenosine in mediating some of the cellular and behavioral responses to ethanol , 40. CafConsidering the alterations in monoamine levels, the results showed that the depression-like behavior produced by ethanol in the behavioral tests was followed by a decrease in norepinephrine and dopamine content in the prefrontal cortex, and this effect was reversed by aminophylline treatment, only in the lower dose.According to the monoamine theory of depression, depressive disorders could be a result of low concentrations of monoamines such as norepinephrine, dopamine, and serotonin in putative brain areas , 45. The2A/dopamine D2 and adenosine A1/dopamine D1 receptors is at least partly responsible for the motor stimulant effects of adenosine receptor antagonists such as caffeine, and for the motor depressant actions of adenosine receptor agonists [Adenosine is an important neuromodulator of the CNS. In fact, this purine nucleoside can modulate a variety of neurotransmitters such as norepinephrine, dopamine, and serotonin . The modagonists . Dopaminagonists . AccordiRegarding the metabolites analyzed, we observed that DOPAC levels, the main dopamine metabolite in rodents, were increased after ethanol administration. A similar finding was presented by Myung et al.  in a stuEthanol produced a depression-like behavior as well as decreased the levels of NE and DA in the prefrontal cortex of mice, and these effects were reversed by the administration of aminophylline, a non-selective adenosine receptor antagonist.


EHP, In the January 2011 issue of In experiments in Swiss mice using low-level lead exposures similar to and lower than those used by In any case, we agree with


This Editorial announces a new section in the Journal of Translational Medicine: Patient-Targeted Molecular Therapies. This section is dedicated to the dissemination of targeted molecular therapies in context of patient-centered outcomes research and evidence-based clinical decisions. The focus on patient-targeted molecular therapies – spanning small molecules and biomolecules alike – stems from the unprecedented growth in this arena. This is consonant with the overall objective of the Journal of Translational Medicine, which seeks out to expand firmly to other vast areas of medicine in the domain of translational science, viewed here as the transaction between translational research and translational effectiveness. As we inaugurate this new section in Journal of Translational Medicine, with its mission described in detail in this Editorial, we invite interested scientists to submit their work for publication. Analyses of current limitations and deficiencies in health care in the context of the Affordable care Act, signed into law by President Obama in March 2010 [There has been unparalleled and continuous progress in research and development in broad areas of molecular medicine resulting in novel platform technologies, drugs, biomolecules and companion diagnostics on the market and in development. Additionally, post-approval testing of novel medicines or therapeutic combinatorial modalities with the aim to improve the standard of care has been a steady continuous source of information with impact to the healthcare. Key to these advances is the dynamism of this process, and the magnitude and complexity of resulting informational output, warranting iterative application of appropriate research and evaluation tools across entire domains of medicine, to identify and promote standard of care or outline state of the art.Therefore, innovative models of evidence-based patient-centered molecular-targeted interventions are timely and critical. Evidence-based medicine/dentistry/nursing, through the process of comparative efficacy and effectiveness research and review for clinical practice (CEERAP) pursues the identification of the best available evidence gained from the scientific method to clinical decision-making ,3. CEERAPatient-Targeted Molecular Therapies, which is dedicated to the dissemination of targeted molecular therapies in the context of patient-centered outcomes research and evidence-based clinical decisions. Therefore, the focus on patient-targeted molecular therapies – spanning small molecules and biomolecules alike – stems from the unprecedented growth in this arena, fueled by scientific progress in understanding the molecular pathogenesis of disease.In response to this urgency, the Journal of Translational Medicine now inaugurates a new section, entitled Patient-Targeted Molecular Therapies provides avenues for the discussion of translational effectiveness pertaining to diverse therapeutic areas including inflammation, degenerative disorders, cancer, infection, metabolism, neurologic, cardiovascular to genetic disorders. The section considers pathologies that pertain to the micro-environment and local immunoregulation and epigenetics, as well as systems immunophysiology. The section addresses chronic and acute immune-related pathologies, from autoimmune disease, to HIV/AIDS, from immune deficiencies to osteo-immunopathology, from inborn errors of immune metabolism to psycho-neuroendocrine-immune pathologies. This section also considers non-immunological disorders, and conditions that are at the interface with immunopathology with both facets: the drug (or therapeutic intervention) and the companion biomarkers.The section Patient-Targeted Molecular Therapies will be complemented by an interest in prognostic and predictive biomarkers, as well as companion diagnostics as they are a critical tool in the assessment or application of molecular targeted therapeutics, and emphasize patient-targeted research for personalized medicine, now subsumed under the terminology “patient-centered outcomes research”. As molecular targeted therapies – through an increased potential for clinical benefit and data-rich clinical design - carry the benefit of yielding informative data sets in individual patients during smaller trials in early development, a focus will be to outline emerging evidence of efficacy or toxicity for innovative drug candidates.The purpose of this new section is consonant with the overall objective of the Journal of Translational Medicine, which – with traditional and diligent interest in immune therapies - seeks out to expand firmly to other vast areas of medicine. This section on Patient-Targeted Molecular Therapies in Journal of Translational Medicine specifically addresses the best available evidence about the translational effectiveness of biomarkers-guided targeted molecular therapies of established medical and dental clinical conditions.In brief, as the informational output regarding more mature or approved molecular targeted therapies evolves across multiple development programs and studies, the specific content of this section will include research products in the form of systematic reviews, clinically relevant complex systematic reviews and meta-analyses. It will also include reviews of the best available evidence, in the form of critical evidence reviews and evidence-based policies. The scientific focus of this section on With this opening editorial, we announce and inaugurate this new section in Journal of Translational Medicine. We invite interested scientists to submit their work for publication.


Trafimow  rejects Consider the active debate on the contributions of fMRI to cognitive theory (e.g., Mather et al., Both this argument and Trafimow's  more genThat said, it is true that no evidence can be offered regarding existing theories if they all assign equal likelihood to all outcomes, or if they cannot be subject to model comparison. In practice, such a situation might less often reflect sloppy inference than a failure to adequately articulate, develop or embellish a theory.


Vibrio. The Gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence.Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus  Vibrio, and of all food-borne pathogens, the US Centers for Disease Control reported that only infections by those of this genus increased (by 78%) between 1996 and 2006 Vibrio vulnificus V. vulnificus is part of the normal bacterial flora of estuarine waters and occurs in high numbers in molluscan shellfish around the world V. vulnificus cases reported in the USA from 1988–2010, with over 500 associated fatalities . Infections occur rapidly, with median incubation times to onset of symptoms being as little as 7 hours V. vulnificus endotoxin by estrogen V. vulnificus, and we would expect to see a far greater number of cases than are typically reported each year. The question then arises as to why so few of these infections are reported each year in the USA.Of all seafood-associated pathogens, none are as critical as those of the genus V. vulnificus virulence and the molecular basis of its interaction with human and oyster hosts is the key to this question. Despite a high degree of phenotypic and genotypic heterogeneity among V. vulnificus strains all known putative virulence determinants have been found to be expressed in both clinical and environmental isolates 50 ≤103), but only 31% of 13 E-genotype strains in an iron-overload mouse model .Understanding the mechanism of V. vulnificus strains based on 16S rDNA gene polymorphism, and Nilsson et al. V. parahaemolyticus from non-pandemic strains. Using the same strategy, we identified an approximately 200 bp RAPD-PCR amplicon significantly associated with clinical isolates vcg (termed the Virulence Correlated Gene) led to a PCR-based assay that can separate V. vulnificus into two groups which strongly correlate to the source  of their isolation in situ detection of C-genotype V. vulnificus strains present in raw oysters. These two genotypes may in fact be different ecotypes, as the genetic dimorphisms are not limited to the vcg gene, but occur throughout the chromosome and appear to dictate the species' environmental preference V. vulnificus strains, including three E-strains and ATCC 33149 ab initio, but must be mapped to the C reference genomes. This approach left the possibility that regions of the E genome for which there is no reference in the C sequence remained undetected. In the present study, we report on the sequencing of three strains of the E-genotype of V. vulnificus, using Roche 454 GS Titanium sequencing. Genomes have been assembled ab initio into large contigs, and the genomic sequences are estimated to be over 99% complete. These newly sequenced genomes have been compared to three previously published C-genotype genomes, strains CMCP6, YJ016, and MO6-24/O. The results of our comparison indicate several significant differences in gene content between the C- and E-genotypes of this pathogen, including genomic regions unique to the E-genotypes, which provide initial insights into the functional basis of pathogenicity in V. vulnificus.Several approaches have been used to identify genotypic factors that distinguish between virulent and avirulent isolates of this pathogen. Aznar et al. V. vulnificus strain JY1305. Given the known sizes and expected variability of V. vulnificus genomes, we estimated that this is equivalent to ∼33× coverage depth of the V. vulnificus JY1305 genome, of estimated size 5.7 Mb. We obtained 671,521 reads of average length 281 bp. The data were assembled into 159 large contigs and 9,184 unassembled fragments using the MIRA assembler, version 3.0 V. vulnificus CMCP6 genome V. vulnificus CMCP6 was recently re-annotated and is regarded as the most complete and accurate of the published V. vulnificus clinical strain genomes http://gibas-research.uncc.edu.188,710,063 nucleotide bases were generated for V. vulnificus JY1305 is composed of 2 circular chromosomes with an estimated total of approximately 5.7 Mb of genomic DNA. V. vulnificus E64MW is estimated to be nearly identical in size, with V. vulnificus JY1701 slightly smaller at 5.6 Mb. Some Vibrio strains are known to have plasmids, but the V. vulnificus JY1305 sequence data contained no evidence of extrachromosomal DNA. PCR validation was performed to verify this finding and no plasmid DNA was found in the genomic DNA preps. It is unknown if V. vulnificus E64MW and V. vulnificus JY1701 contain plasmid DNA, but no plasmid sequence with homology to known V. vulnificus YJ016 plasmid sequences was identified, either in the assembled genomic sequence, or among the unassembled reads.The genome of V. vulnificus strains were co-analyzed with genome sequences of strains CMCP6, YJ016, and MO6-24/O to identify LCBs common to C and E strains. The V. vulnificus CMCP6 genome was used as the reference genome in this analysis. At a size threshold of 1% of the genome, or 57 kb or greater, there are a total of 24 locally collinear sequence blocks that are conserved in the six V.vulnificus genomes. All of these large blocks are found in each of the six V. vulnificus strains, and they cover approximately 68.5% of the genome. At a size threshold of 90 aa (270 bp) or greater, we find an additional 186 LCBs. At this size threshold, LCBs are not necessarily conserved across all six genomes, and may correspond to individual genes or genomic islands that differentiate among the sequenced strains. V. vulnificus strains  used in this study. V. vulnificus genomes, and LCBs  are defined as conserved segments that appear to be internally free from genome rearrangements relative to the other genomes in the set under study Vibrio vulnificus genomes as described in V. vulnificus genomes as well as to a broader group of all 16 previously completely sequenced genomes belonging to the genus Vibrio , which is commonly used in combination with other markers to distinguish V. vulnificus from other Vibrio spp. in molecular assays, were found, as expected, in all six V. vulnificus strains, which gives us confidence in the sequencing and differential analysis. A related gene, VVA0964, the cytolysin secretion protein gene vvhBV. vulnificus genomes and may have potential as a diagnostic marker. The gene encoding zinc metalloprotease, VV2-0032 (vvpE), another commonly-used diagnostic marker, was identified by Gulig et al. 2010 as being common to both E-genotypes and C-genotypes We identified 1748 core genes that are common to the three draft E-genotype genomes and the 16 V. vulnificus genes are the Flp pilus genes. We believe this is a novel observation, as we have not seen it discussed elsewhere. The E- and C-genotypes of V. vulnificus contain a nearly identical operon for the assembly of an Flp pilus, a type IV pilus that mediates adherence, including genes for Flp pilus assembly CpaB, CpaC, a conserved unknown protein, and CpaE. The Tad assembly proteins of the Flp pilus, including TadA, TadB, TadC, and TadD, are also highly conserved and identically ordered in C7184 and YJ016. Both E- and C- strains of V. vulnificus contain all the components of the Tad assembly proteins except TadD, while other Vibrio spp. do not. These genes may be part of a tad (tight adherence) locus, found in a wide variety of bacteria, that is characteristic of horizontal gene transfer. tad loci are generally present as part of a mobile genetic element, specifically the “widespread colonization island” tad loci are proposed to facilitate environmental niche colonization Also found in the list of 940 core V. vulnificus  V. vulnificus. The differential functional analysis shows that GO terms mannitol-1-phosphate 5-dehydrogenase and N-acetylneuraminidase are significantly enriched in the C-types with an adjusted p-values of 2.42E-04 and 1.13E-05, respectively. Specifically, 35% of the genes associated with mannitol-1-phosphate 5-dehydrogenase activity and nearly100% of the genes with associated N-acetylneuraminidase function are found to be unique to C-types. Additionally, GO terms “chondroitin AC lyase activity” and “arylsulfatase activity” are significantly enriched with adjusted p-values of 0.0068 and 0.048, respectively. In both categories, close to 100% of the genes are only found in the C-genotype differentials. In contrast, the E-genotypes appear to be strongly enriched in genes associated with the GO functions “urea metabolic process” and “nickel ion binding”. Nearly all of the genes that fall under these GO categories are only found in the E-genotypes. Both show up as statistically significant differentials with adjusted p-values of 1.52E-09and 4.37E-07, respectively. Additionally, E-genotypes appears to have several unique genes that fall into GO categories associated with carbohydrate transport and transmembrane transporter activity for a variety of sugars and sugar derivatives.Understanding the overall significance of these genotypic GO functional differences will require further investigation, however we propose that these differentiating functional categories may be relevant to the SPANC hypothesis which describes the balance between self-preservation and nutritional competence in bacterial genomes Vibrionaceae family may play a role in adaptation to environmental changes V. vulnificus MO6-24/O . Based on the location of the E-genotype differentiating genes in the LCB alignments  balance, could potentially offer insight into the niche adaptation and differentiation seen in V. vulnificus C- and E-genotypes. The SPANC hypothesis has been well characterized in E. coli and demonstrates that clonal populations can experience genetic mutations and phenotypic changes as a result of physiological stress under conditions such as nutrient starvation. These changes often lead to variations in the activity of the global gene regulator, sigma factor σS (rpoS), which governs the general stress response. Decreased RpoS activity can lead to the development of specialized populations which are less resistant to stress but have broader nutritional capabilities and a higher affinity for low nutrient concentrations, whereas the original population is more stress tolerant but less nutritionally competent As previously mentioned, V. vulnificus species. By completely sequencing three E-genotypes of V. vulnificus, we were able to examine what genes are unique to E-genotypes. As noted above, the GO functional gene content differences between C- and E-genotypes showed that the sequenced E genomes have significant enrichment for genes associated with metabolic functions such as urea and nitrogen cycle metabolism . In addition, both C- and E-genotypes are enriched for GO functions associated with transmembrane transport of various organic compounds (It seems plausible that this trade-off between self-preservation (stress resistance) and nutritional competence could be a factor driving the diversification of tabolism , suggestompounds . Indeed,Alteromonadales TW-7, with a significant blast hit . Using site directed mutagenesis of conserved aromatic residues within the PKD domain, Orikoshi et al.Alteromonas sp. strain O-7 was required for effective binding and hydrolysis of chitin. As noted by Grimes et al.V. vulnificus (CMCP6 and YJ016). However, the PKD gene found in the newly sequenced E strains may be a chitinase that is unique to E-genotypes. The ability to attach to chitin is important for Vibrio spp. as it facilitates DNA transformation, believed to be critical for horizontal gene transfer in this genus In the E-genotypes, we also identified genes associated with specific attachment proteins which would likely facilitate environmental survival. Polycystic kidney disease I domain (PKD) is a unique domain that can be found within chitinases, which has been proposed to enhance the hydrolysis of insoluble chitin V. vulnificus to survive stressful conditions have shown that C-genotypes are significantly better able to survive in complement-activated human serum than E-genotypes 202 exposure or elevated temperature Vibrio vulnificus, in that C-genotypes are more capable at self-preservation, while E-genotypes carry additional genes that suggest they may be more capable of nutritional competence. Sequence alignments of the rpoS gene for all six sequence strains did not indicate any major genetic polymorphisms, and only resulted in a few amino acid substitutions. The nucleic acid sequence is ∼99% identical and the coded protein 98.5% identical. Other genes that may affect the SPANC balance The ability to cope with the rapid and potentially stressful transition from the oyster environment to the human host likely requires a variety of stress resistance genes that provide the bacterium with protection and the ability to survive in this seemingly hostile environment. Previous studies have demonstrated the need for stress regulators to aid in survival under a variety of stressful conditions, such as starvation, osmotic stress, low pH, non-optimal temperatures, and oxidative damage vcgC and 16S rDNA type B) Mannitol transport and fermentation genes were found to be present in the C-genotype strains but not in the newly sequenced E-genotype strains. Mannitol has been correlated with virulence-associated genotypes  used MLST data to identify a 33-kb genomic island (region XII) on the second chromosome of V. vulnificus E. coli, they facilitate invasion of the blood-brain barrier V. vulnificusArylsulfatases occur in virtually all organisms, and are found in high levels in the digestive glands of oysters and other mollusks V. vulnificus adheres and begins its infective route, and previous studies have demonstrated the importance of this gene for growth, adhesion, and survival within the jejunum and colon tissues of the mouse intestine nanA, nanE, and nanK) is responsible for sialic acid and catabolism has been identified in several major intestinal pathogens including V. vulnificusnanA. However, recent work in our lab  and others investigating the presence of nanA in a larger number of clinical and environmental genotypes has revealed the presence of this gene in some E-genotypes nanA gene is less prevalent in E-genotypes and there also appears to be some correlation between the presence of this gene and C-genotypes, highlighting the need for further investigation into the function of sialic acid catabolism as a virulence factor for V. vulnificus.Sialic acids are a family of nine-carbon sugar acids that are typically located at the terminal carbohydrate ends of mucin proteins. The most abundant sialic acid is N-acetylneuraminic acid (Neu5Ac), with all of the other sialic acids being derivatives of this compound V. vulnificus, stressful conditions such as carbon starvation, non-permissive pH values, and hyperosmolarity will induce a stress response in which sigma S (σS) competes with the housekeeping sigma factor (σD) thus redirecting gene expression to respond to the stress. Gram positive bacteria, such as Bacillus subtilis and Staphylococcus aureus, use a comparable alternative sigma factor (σB) which governs the “general stress response” and plays an important role in virulence in these organisms. This is accomplished by activating the transcription of over 125 genes in response to a variety of stressful conditions, including temperature shifts, ethanol, salt and acid stress, and starvation B. subtilis, σB activation is partly regulated by a large signaling complex called the RsbRST stress module (or stressosome), and a PP2C-type phosphatase, RsbU A hallmark method for responding and adapting to environmental fluctuations involves the use of alternate sigma factors which compete for RNA polymerase and subsequently initiate the transcription of a specific subset of genes rsbR, which we found in E64MW and JY1701, this stressosome was absent in the E-genotypes we sequenced. To our knowledge, V. vulnificus does not possess the σB subunit of RNA polymerase, thus the role of this signaling system in V. vulnificus is not clear. It has been proposed by some investigators that the function of these modules may vary considerably amongst bacteria as a result of niche expansion in which incoming signals are relayed to a diverse array of regulatory systems, such as alternative sigma factors and two-component signal transducing systems V. vulnificus genotype. Additionally, elucidating the role of this system in V. vulnificus would be of great interest and could potentially provide insight into the mechanisms of virulence and survival in this organism.In all three C- genotypes, we identified an operon homologous to the RsbRST stress response module, the PP2C-type phosphatase, and a downstream two-component regulatory system. With the exception of a single gene encoding Vibrio spp. have been shown to possess a large number of these regulators indicating the importance of cyclic-di-GMP signaling in this genus in their adaptation to new environments E. coli, yeaJ is one of the many GGDEF domain encoding genes that differentially mediates switching between motility and curli-fimbrial mediated adhesion Cyclic-di-GMP is an intracellular signaling molecule that acts as a second messenger for integrating environmental signals and has been demonstrated to regulate several distinct cellular processes such as motility, biofilm formation, virulence, and rugose colony morphology Salmonella enterica serovar Typhimurium, FimZ acts as a positive transcriptional activator of type I fimbrial expression, therefore modulating ability to attach or swim in a given environment FimZ results in a lack of motility due to the down-regulation of the flhDC master flagellar operon. Clegg's group has also shown that FimZ plays a crucial role in regulating the expression of phenotypes associated with adherence to, and invasion of, eukaryotic epithelial cells In other pathogens, such as VirB4 (VV2_0638) was found to be present in C-genotype strains (V. vulnificus YJ016 and V. vulnificus CMCP6) but absent in the newly sequenced E-genotype isolates and V. vulnificus MO6-24/O. Type IV bacterial secretion systems (T4SS) are responsible for the translocation of molecules such as DNA, proteins, and toxins out of the cell and into the immediate environment or the host cell VirD4) VirB4 serve as energizing components as these genes are associated with ATPase functionality V. vulnificus E-genotype strain sequencing suggests that these T4SS components are active in infections caused by C-genotypes (V. vulnificus YJ016 and V. vulnificus CMCP6). 70% of the predicted virB operon sequence of the T4SS has been observed to be present in the C-genotypes (V. vulnificus YJ016 and V. vulnificus CMCP6) and not in M06-24 or the E-genotypes Vibrio vulnificus.Type IV secretory system gene Vibrio vulnificus have been sequenced to over 99% completion. The genomes have been assembled using ab initio methods and contig sequences have been deposited in the NCBI Whole Genome Shotgun archive. Additional Illumina sequencing is underway with the aim of complete closure of the strain JY1305 genome. We expect that effort to provide insights into structural rearrangements among the C-genotype and E-genotype strains, but we do not expect the additional sequencing to significantly alter the findings of strain-differentiating genes reported herein. Current work in progress also includes the genomic sequencing of a larger collection of V. vulnificus strains, encompassing the entire genomic spectrum of pathogenicity. That data will provide additional insights into the distinct genomic differences between pathogenic and non-pathogenic strains . The comparative analysis of C- and E- genotypes confirms previous observations of putative virulence determinants that differentiate V. vulnificus isolated from wounds in clinical settings from environmental strains. However, the analysis also points the way to dozens of differentiating genes specific to the E-genotypes. Some of the genes potentially fit into existing functional hypotheses such as the SPANC theory, while others are as yet functionally uncharacterized.In conclusion, three E-genotype strains of V. vulnificus community with a valuable reference for functional study of determinants of virulence, and facilitates the future use of high-throughput approaches to assess functional differences via study of the V. vulnificus transcriptome. Future studies will aim to analyze gene locations and gene neighborhoods to determine if there are genotypic differences here that could account for differences in physiology; e.g. our mannitol study revealed differences in the gene arrangement of a putative hemolysin, mannitol transporter, and mannitol fermentation operon that has been shown to correlate with clinical C-genotypes Although the presence or absence of a particular gene in a specific genotype provides initial targets for functional differentiation, this current sequencing effort provides the V. vulnificus strain JY1305 was grown overnight in Bacto™ Heart Infusion (HI) broth  at 30°C with vigorous shaking. Cells were pelleted by centrifugation and supernatants discarded. The cells were washed three times with phosphate buffered saline (PBS) before being resuspended to a final approximate concentration of 5×108 cell/ml. The MagMax™ Total Nucleic Acid Isolation Kit (Ambion) and All Prep DNA/RNA/Protein Mini Kit (Qiagen) were used for DNA extraction. The quality and quantity of DNA was evaluated spectrophotometrically with the NanoDrop ND1000 . A concentration of 50 ng/µL was used for next gen sequencing.V. vulnificus strains JY1701 and E64MW were grown overnight with shaking in 10 ml of ASPW. Cells were pelleted by centrifugation and resuspended in 100 µl of ice-cold PBS. DNA was extracted using DNAzol (Invitrogen) according to manufacturer instructions, followed by incubation with RNase A. Subsequently, samples were purified using a phenol/chloroform/isoamyl alcohol extraction protocol. Briefly, 40 µl of 3 M sodium acetate was added to each DNA sample, followed by 440 µl of phenol/chloroform/isoamyl alcohol. Samples were centrifuged  and ∼400 µl of supernatant was removed and mixed with an equal volume of phenol/chloroform/isoamyl alcohol. This solution was centrifuged  and the supernatant (∼300 µl) removed and mixed with an equal volume of 24∶1 chloroform/isoamyl alcohol. Samples were subsequently centrifuged for 5 min  and the supernatant (∼200 µl) was subjected to ethanol precipitation. The DNA pellet was re-dissolved in 50 µl 1×TE buffer and stored at −80°C. The quality and quantity of DNA was subsequently ascertained spectrophotometrically using a NanoDrop ND1000 .V. vulnificus JY1305 was sequenced at the Virginia Commonwealth University using Roche/454 Titanium technology V. vulnificus E64MW and JY1701 were sequenced at the BBSRC Genome Analysis Centre  also using the Roche/454 Titanium technology V. vulnificus JY1305, E64MW and JY1701, were aligned to three V. vulnificus C strain reference sequences  and longest common blocks (LCBs) were identified in each genome. V. vulnificus YJ016 and MO6-24/O and each set of assembled contigs for JY1305, E64MW, and JY1701 were aligned against the reference sequence (V.vulnificus CMCP6) to produce separate, optimal pair-wise alignments for each query sequence. The pair-wise alignments were then used to produce a multiple alignment of the newly sequenced strains, and V. vulnificus YJ016 and MO6-24/O, using V. vulnificus CMCP6, which has recently been re-annotated Contigs from each assembly were aligned to reference genomes using the Mauve software V. vulnificus strains, was absent in the newly sequenced E-strain genomes csrA F2, csrAR2, rpod UP, rpod DOWN, vvhA F, vvhA R, pepRF F2 and pepR3. These were designed based on known features of the C-type genomes. The primers used to test for the presence of a YJ016-type plasmid were vvSSF1, vvSSR1, vvF2 forward primer, and vvR2 reverse primer. Using Primer3 V. vulnificus CMCP6 and YJ016 strains using BLAST to ensure that they exclusively matched the two plasmid sequences. Primer sequences are provided in The LCB alignment results suggested that a plasmid sequence, present in YJ016 and thought to be present in other V. vulnificus reference organisms (CMCP6 and YJ016). Spacer sequence was added to the ends of each contig to mimic start and stop signals. The spacer sequence was 32 nucleotides in length. We used the sequence NNNNNCACACACTTAATTAATTAAGTGTGTGNNNNN, which is used at JCVI to merge contigs [http://www.jcvi.org/cms/research/projects/annotation-service/submission-guide/].Draft annotation of the sequences was performed using a pipeline of published microbial annotation tools. Feature determination for each strain was performed on the contig set from each sequence assembly. The feature identification methods that were used were Glimmer3.02 and GeneMark.hmm Vibrio spp. The first two criteria were derived from Chen et al., 2003 For gene identification in each of the newly sequenced strains, one of the following criteria had to be met: (1) A gene will be included in the gene list if it can be predicted by either Glimmer or GeneMark, as long as it amino acid sequence length is equal to or greater than 150. (2) A gene must be predicted by both Glimmer and GeneMark to be included in the gene list, if its amino acid sequence length less than 150. (3) A gene prediction will only be included in the gene list if it occurs in a cluster of known or hypothetical genes found in other tRNAScanSE was used to predicted the tRNAs in the MIRA contigs for each strain Vibrio genomes GenBank identifiers .The reference genomes used for the comparative genomic content analysis include all the available and completely characterized Vibrio spp. V. vulnificus C-type strains, and then with the 16 fully sequenced Vibrio species, to determine the impact of different reference sets on the orthology analysis outcome.OrthoMCL version 2.0 was used to cluster newly predicted genes with genes from other Vibrio spp.The OrthoMCL clustering generated during the annotation step was used as the basis for identification of differentiating genes. Identified gene features and OrthoMCL results were stored in a locally developed OLAP data warehouse (GenoSets) that supports queries across aggregate data generated by a variety of genomic annotation and comparison methods. This system is fully described in  V. vulnificus strains were associated with GO terms using homology determined through OrthoMCL clustering of BLASTP results. For functional comparison purposes, it is helpful to identify genes and other features using a controlled vocabulary. The Gene Ontology (GO) provides standardized terms for the description of gene products in terms of biological processes, cellular location, and molecular function In order to provide a standard means of comparison for feature attributes we establish relationships between features using two methods. First, we estimate orthologous relationships between genes using OrthoMCL, which uses a Markov Cluster algorithm to group putative homologs based on sequence similarity, as the primary ortholog clustering method in GenoSets. OrthoMCL has been shown to outperform other stand-alone methods for ortholog clustering Vibrio spp. We performed a phylogenetic analysis following the methods used in Suzuki et al. and Hasan et al. We identified 1748 single-copy ortholog clusters within 19 Table S1V. vulnificus genomes.Conservation of locally collinear blocks (LCBs) in (DOCX)Click here for additional data file.Table S2V. vulnificus and other Vibrio spp.Summary of key gene differences between (DOCX)Click here for additional data file.Table S3Primer sequences used to validate presence of genomic DNA and plasmid DNA in extracted samples.(DOCX)Click here for additional data file.Supplement S1V. vulnificus JY1305, E64MW, and JY1701 and alignment position in the V. vulnificus CMCP6 reference genome.Complete list of predicted genes for the draft assemblies of (XLSX)Click here for additional data file.Supplement S2Complete listing of LCBs  identified by Mauve among the newly sequenced E-genotypes and the C-genotype reference strains.(XLSX)Click here for additional data file.Supplement S3V. vulnificus from other Vibrio spp.Complete list of genes that differentiate (XLSX)Click here for additional data file.Supplement S4Complete list of genes differentiating E-genotype strains from C-genotype strains.(XLSX)Click here for additional data file.Supplement S5Details that support the rationale for aspects of the bioinformatic analysis.(PDF)Click here for additional data file.


Environmental Health Perspectives (EHP) established the Paper of the Year Award in 2008 to recognize high-quality papers published in the journal (EHP decided to recognize two papers each year (EHP Classic Paper of the Year is the research article, commentary, or review that is the most highly cited over the preceding 60 months. The winner of the 2012 EHP Classic Paper of the Year, announced last July, was “Exposure of the U.S. Population to Bisphenol A and 4-tertiary-Octylphenol: 2003–2004” by Antonia M. Calafat, Xiaoyun Ye, Lee-Yang Wong, John A. Reidy, and the late Larry L. Needham of the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention (EHP Paper of the Year. This award recognizes the most highly cited paper published during the preceding year. Both awards are subject to the approval of the EHP Board of Associate Editors. journal . Startinach year . The EHPevention . The secEHP is pleased to announce that the 2012 Paper of the Year award will be shared by three papers published in the same issue: “Prenatal Exposure to Organophosphates, Paraoxonase 1, and Cognitive Development in Childhood” by Stephanie M. Engel, James Wetmur, Jia Chen, Chenbo Zhu, Dana Boyd Barr, Richard L. Canfield, and Mary S. Wolff (In this issue, S. Wolff ; “PrenatS. Wolff ; and “SeS. Wolff . The thrS. Wolff ; the UniS. Wolff ; and theS. Wolff . The BerEHP congratulates all the authors of these papers for their contribution to the environmental health science literature. These findings underscore the need to continue monitoring levels of exposure to pesticides in vulnerable populations and to study the under-lying biological changes associated with alterations of cognitive function following developmental exposure to environmental chemicals.


Parkinson's disease is a common neurodegenerative disease characterised by progressive loss of dopaminergic neurons, leading to dopamine depletion in the striatum. Mutations in the PINK1 gene cause an autosomal recessive form of Parkinson's disease. Loss of PINK1 function causes mitochondrial dysfunction, increased reactive oxygen species production and calcium dysregulation, which increases susceptibility to neuronal death in Parkinson's disease. The basis of neuronal vulnerability to dopamine in Parkinson's disease is not well understood.We investigated the mechanism of dopamine induced cell death in transgenic PINK1 knockout mouse neurons. We show that dopamine results in mitochondrial depolarisation caused by mitochondrial permeability transition pore (mPTP) opening. Dopamine-induced mPTP opening is dependent on a complex of reactive oxygen species production and calcium signalling. Dopamine-induced mPTP opening, and dopamine-induced cell death, could be prevented by inhibition of reactive oxygen species production, by provision of respiratory chain substrates, and by alteration in calcium signalling.These data demonstrate the mechanism of dopamine toxicity in PINK1 deficient neurons, and suggest potential therapeutic strategies for neuroprotection in Parkinson's disease. Mitochondrial dysfunction plays a major role in the pathogenesis of Parkinson's disease (PD), and has been demonstrated in mendelian PD models, toxin based PD models, and studies of sporadic PD brain tissue Although this mitochondrial pathophysiology exists in all neurons in the brain, neuronal death in Parkinson's disease is specific for certain brain regions. In the early stages of sporadic Parkinson's disease, one of the pathological hallmarks is the loss of substantia nigra pars compacta (SNpc) dopaminergic neurons, although as the disease progresses, non-dopaminergic neurons eventually become affected. Indeed, the initial selectivity of dopaminergic neurons remains a fundamental question in PD biology. Dopaminergic neurons are neurons that synthesise, package and release dopamine, and are thus exposed to intracellular and extracellular dopamine. Therefore it has been suggested that dopamine itself may be the cause of the selective cellular vulnerability in PD. However the interaction between mitochondrial dysfunction and sensitivity to dopamine has not yet been shown in genetic models of PD, and therefore it is unclear how mitochondrial dysfunction may particularly render dopaminergic neurons vulnerable to cell death.In this study we have investigated the effect of dopamine in a model of mitochondrial dysfunction in PD induced by PINK1 deficiency. We have previously reported that dopamine induces a cytosolic calcium signal in astrocytes and neurons through receptor- independent mechanisms 2+]c and mitochondrial membrane potential (Δψm) in postnatal midbrain co-cultures of astrocytes and neurons from wildtype (wt) and PINK1 knockout (ko) mice. In control cells, application of 20 µM dopamine induced small but significant sporadic changes in [Ca2+]c . This was not associated with any change in Δψm  was prevented by pre-incubation with a PTP inhibitor, Cyclosporin A (0.5 µM) ; fluo-4 signal rose by 750±80 arb.U; n = 127 astrocytes). In wt neurons and astrocytes there were non significant changes in the Rhod-5n fluorescence ([Ca2+]m).Cells were loaded with Rhod-5n and fluo-4 to simultaneously measure [Ca2+]m appeared higher in PINK1 ko neurons than in wt neurons  which were associated with an increase in [Ca2+]m, which eventually led to a rapid decrease of mitochondrial rhod-5n signal . This is due to the opening of the PTP, as it was prevented by incubation with CsA. 2+]c and [Ca2+]m changes .Previously we demonstrated that PINK1 deficiency is associated with increased basal production of ROS in the mitochondria and cytosol (mediated by NADPH oxidase). We therefore inhibited basal ROS generated by NADPH oxidase using diphenylene iodonium  for 20 minutes prior to these experiments in order to investigate the specific effects of dopamine. Application of 50 µM dopamine to wt and PINK1 ko neurons in the presence of 0.5 µM DPI produced a profound activation of ROS production in both wt and PINK1 ko neurons  respiratory chain in mitochondria (2) NADPH oxidase and (3) MAO. We tested the effects of inhibiting each source of free radicals on dopamine induced mitochondrial depolarisation in PINK1 ko neurons. Pre-incubation of cells with the mitochondrially localised antioxidant MitoQ (10 µM) prevented the dopamine induced mitochondrial depolarisation in PINK1 ko neurons . We next investigated whether the compounds that had been shown to reduce dopamine induced mitochondrial depolarisation had any effect on dopamine induced cell death. Of note, pre-incubation of cells with the inhibitor of PTP, 1 µM CsA, significantly reduced the number of dead cells in PINK1 co-cultures  high cytosolic levels of calcium, resulting in mitochondrial calcium overload and opening of the PTP, and (2) oxidative stress 3 and a Ca2+ signal We have demonstrated that in PINK1 deficient cells, dopamine is able to induce opening of the PTP through two major processes that may act synergistically: calcium signalling and ROS production. Dopamine is able to induce a calcium signal in neurons and astrocytes by receptor dependent as well as receptor independent mechanisms. In astrocytes, dopamine induces a calcium signal by MAO-dependent metabolism producing hydrogen peroxide, which activates lipid peroxidation resulting in activation of phospholipase C, release of IP2 signal is not associated with significant changes in mitochondrial Ca2+. However in PINK1 deficient cells dopamine induces a mitochondrial Ca2+ signal, which may occur with or without concomitant visual changes in the cytosolic Ca2+. In neurons and astrocytes mitochondria act as the major buffer for cytosolic Ca2+, and exist in close relation either to the potential dependent calcium channels, or to the endoplasmic reticulum. Such microdomains result in alterations in calcium signal being visualised in single compartments within the cell, such as the mitochondria but not necessarily in the cytosol 2+ due to an impairment of mitochondrial calcium efflux 2+ that is sufficient to induce opening of the PTP and cause mitochondrial membrane depolarisation. This dopamine-induced mitochondrial depolarisation can be prevented by the use of calcium chelation in both neurons and astrocytes. Thus the inherent mitochondrial calcium dysregulation caused by loss of PINK1 function, renders neurons particularly vulnerable to repeated dopamine induced calcium elevations.In both wildtype and PINK1 deficient cells, we have confirmed that dopamine induces a receptor independent calcium signal i.e the calcium signal is MAO dependent in astrocytes, and VDCC dependent in neurons. Importantly, in wildtype cells, this dopamine induced cytosolic CaPTP opening is also regulated by the redox state of mitochondria. We have demonstrated that dopamine induces a change in ROS balance in control cells and PINK1 deficient cells. The mechanism of ROS production in PINK1 deficient cells is three-fold: (1) activation of NADPH oxidase and (2) respiratory chain deficiency both occur in the basal PINK1 deficient state, and (3) MAO activity in metabolism of dopamine. Preventing ROS production through each mechanism is able to prevent the dopamine-induced PTP opening in PINK1 deficient neurons, proving that dopamine induced ROS production is sufficient to induce PTP opening in vulnerable neurons. An increase in Δψm leads to an increase in the threshold of PTP opening It is well recognized that there is considerable interplay and reciprocal interaction between calcium and ROS balance within the mitochondria in both signaling and disease. Therefore dissecting the relative importance of each in dopamine induced PTP opening is challenging. We have previously established that an increase in calcium activates NADPH oxidase with a resultant increase in ROS in PINK1 deficient cells Our findings have several implications on therapy in PD. First they raise the controversial question whether exogenous dopamine administration in the form of levodopa therapy may contribute to neuronal toxicity. Dopamine has been known to be toxic to neurons in vitro but evidence of dopamine or L-dopa toxicity in vivo models is lacking, and administration of L-dopa to normal rodents or primates does not result in oxidative damage or nigral cell degeneration. Clinical studies in PD patients have also failed to demonstrate an effect of l-dopa toxicity. However imaging studies accompanying clinical trials have suggested that there is a higher rate of decline in ligand uptake in patients on L-dopa compared to placebo  which raise the possibility that L-dopa administration accelerates cell death This study has further enabled us to deduce novel intervention strategies for neuroprotection in PD. Reduction of oxidative stress within mitochondria using mitochondrial directed antioxidants has been suggested as a therapeutic strategy and this data lends support that it may reduce cell death. However we have found that optimisation of the mitochondrial respiratory function in PINK1 deficient neurons is also highly efficient at preventing dopamine-induced cell death in vitro. The provision of respiratory substrates may therefore represent an intervention that needs to be validated in vivo models of PD. The role of MAO inhibition in PD has been controversial for many years: experimental data has raised the possibility that MAO inhibition may be neuroprotective although the mechanisms remained unclear. We provide evidence that MAO inhibition may prevent dopamine-induced cell death in vitro by limiting ROS production in astrocytes, and by preventing the astrocytic calcium signal induced by dopamine. Finally we have highlighted the role of manipulating neuronal calcium signaling to protect against dopamine induced PTP opening and cell death. Verapamil acts as a calcium channel blocker that reduces the dopamine induced calcium signal in neurons and protects against dopamine induced depolarisation and cell death in susceptible PINK1 deficient neurons.In summary, we have utilised a genetic primary mammalian model of PD to demonstrate the mechanism of dopamine induced neuronal degeneration. Dopaminergic neurons exposed to dopamine over long periods of time will be susceptible to repeated rises in ROS and calcium. Cells that have reduced ability to handle oxidative stress or calcium fluxes, are unable to handle persistent low doses of dopamine which, over time, may become sufficient to induce PTP opening. PINK1 deficient dopaminergic neurons lack the neuroprotective mechanisms to maintain their calcium and ROS homeostasis and are prone to dopamine toxicity. Of note, manipulation of calcium signaling and the redox state of such cells may represent successful strategies to prevent cell death in PD.2 incubator (5% CO2 in air) at 37°C for 3–4 hrs, before replacing the attachment medium with prewarmed maintenance medium . Once the cells were in maintenance medium, half of the medium was replaced weekly. All live cell imaging experiments were performed between d10-d14 in culture.The PINK1 deficient mice were generated by Lexicon Genetics Inc.  (6). For primary mouse midbrain cultures pups were taken at postnatal D2–4. Embryos/pups were obtained either by crossing homozygote animals and comparing same age control animals, or by crossing two heterozygote animals and comparing genotypes within a litter. Animal husbandry and experimental procedures were performed in full compliance with the United Kingdom Animal (Scientific Procedures) Act of 1986. Heads were collected in chilled dissection medium . The whole brain was removed from the skull case in a Petri dish containing chilled dissection medium under sterile conditions, and the meninges were removed. The cortices or midbrain was carefully dissected and transferred to a sterile micro tube containing ∼0.5 ml chilled dissection medium and allowed to settle under gravity. Dissection medium was replaced with 500 µl pre-warmed trypsin solution for 15 minutes at 37°C with gentle agitation halfway through incubation. The trypsination solution was aspirated and the cortices/midbrain washed three times with 500 µl pre-warmed attachment medium . The tissue was triturated using sterile fire-polished glass pipettes of differing pore diameter until a smooth suspension was produced. Cells were placed in a humidified CONeurones were easily distinguishable from glia: they appeared phase bright, had smooth rounded somata and distinct processes, and lay just above the focal plane of the glial layer.2+]c and [Ca2+]m, cells were loaded for 30 min at room temperature with either 5 µM fura-2 AM or fluo-4 AM in combination with either 5 µM Rhod-5n, and 0.005% pluronic acid in a HEPES-buffered salt solution (HBSS) composed of (mM): 156 NaCl, 3 KCl, 2MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose and 10 HEPES; pH adjusted to 7.35 with NaOH. Previous work using direct and indirect approaches confirmed that the mitochondrial calcium concentration is high in PINK1 ko cells and we have found that the low affinity calcium indicator Rhod-5N is more sensitive to detecting mitochondrial calcium changes in these cells than using a high affinity indicator such as XRhod-1 or Rhod-2. Furthermore at the time of PTP opening the high mitochondrial calcium content will result in the fluorescence of all indicators being fully saturated prior to redistribution on membrane disruption (eg PTP opening). We have therefore used Rhod-5N to detect mitochondrial calcium in this study.For measurements of [Ca2+]i and mΔψ, cells were loaded with fura-2 for 30 minutes, and Rh123  was added to the culture during the last 15 min. Cells were washed five times prior to the experiment. Under these loading conditions, Rh123 is non toxic and gives a reliable and reproducible measure of Δψm through the ‘dequench’ of mitochondrial fluorescence.For simultaneous measurement of i and Δψm were monitored in single cells using excitation light provided by a Xenon arc lamp, the beam passing through monochromator centred sequentially at 340, 380 and 490 nm . Emitted fluorescence light was reflected through a 515 nm long-pass filter to a cooled CCD camera . All imaging data was collected and analysed using software from Andor . The fura −2 data has not been calibrated in terms of [Ca2+]i because of the uncertainty arising from the use of different calibration techniques. Accumulation of Rh123 in polarised mitochondria quenches the fluorescent signal; in response to depolarisation the fluorescence signal is dequenched; an increase in Rh123 signal therefore signals mitochondrial depolarisation. Wherever possible, we have normalised the signals between resting level (set to 0) and a maximal signal generated in response to the uncoupler FCCP .Fluorescence measurements were obtained on an epifluorescence inverted microscope equipped with a ×20 fluorite objective. [CaConfocal images were obtained using a Zeiss 510 uv-vis CLSM equipped with a META detection system and a 40× oil immersion objective. The 488 nm Argon laser line was used to excite fluo-4 fluorescence which was measured using a bandpass filter from 505–550 nm. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ∼2 µm. For MitoSOX or Rhod-5N measurements the 543 nm laser line and 560 nm longpass filter were used. For Het, we generated ratios of the oxidised form (ethidium) excited at 543 nm and measured using a 560 nm longpass filter and the reduced form with excitation at 351 nm, measured between 375–470 nm. All data presented were obtained from at least 5 coverslips and 2–3 different cell preparations.Statistical analysis and exponential curve fitting were performed using Origin 8  software. Results are expressed as means ± standard error of the mean (S.E.M.). Paired T-tests were performed for statistical analysis.