Introduction: Investigating the clinical impact of serum uric acid (UA) and its lowering agents on the complications and mortality of acute ST-elevation myocardial infarction (STEMI) can open a new era in STEMI treatment. The aim of this study was to evaluate the effect of on admission serum UA level on the mortality and morbidity of patients admitted with STEMI.Methods: A number of 608 patients with STEMI were enrolled in this study from December 21, 2012 until February 19, 2014. Patients were followed for 20 months. Male to female ratio was 2.53, and the mean age of patients was 62.6±13.4. The relationship between the level of UA and patients’ mortality and morbidity, left ventricular ejection fraction (LVEF), atrial and ventricular arrhythmia was analyzed.Results: Patients with high serum UA level had higher Killip class after STEMI (P=0.001). Mean LVEF was measured to be 39.5±9.6 in normal UA group and 34.6±11.6 in high UA group (P=0.001). In comparison with normal UA group, high UA group had significantly higher cTnI , increased blood pressure (P=0.009), and higher atrial fibrillation (AF) occurrence (P=0.03), but no association was seen between ventricular tachycardia and serum UA level. Short term and midterm mortality were not different in two groups .Conclusion: In the current study, high serum UA level in patients with acute myocardial infarction (MI) was not associated with higher in-hospital or midterm mortality, but it was associated with lower LVEF, higher Killip class, elevated cTnI, creatinine, triglyceride, and higher AF. Irreversible myocardial damage and cell death would occur if occlusion of coronary artery lasts more than 20 minutes.5 Endothelium degrades adenosine to uric acid (UA) rapidly; so due to increasing the concentration of UA and decreasing the intracellular PH, rapid UA efflux to the vascular lumen occurs.6 hence, UA level may be an indicator of severity of ischemia and tissue hypoxia and high serum UA has been stipulated as a risk factor for coronary artery disease (CAD) and somehow as prognostic factor for mortality and morbidity in patients with CAD. Chen et al showed association between serum UA and adverse events in patients with STEMI.7 Kroll et al showed that in patients with higher on admission serum UA, short term and long term mortality was increased.8 In other study done in Iran higher serum UA had association with in-hospital and short term mortality.9 By developing new treatments especially increasing trend to invasive therapy in high risk patients for example those who develop heart failure after ST-elevation myocardial infarction (STEMI) and continuous effort to decrease STEMI mortality, it is interesting to investigate whether prognostic factors such as serum UA continue to have their prognostic value or not.Adenosine produced by cardiac myocytes causes the vasodilatation of arteries. The level of adenosine is increased under some conditions such as hypoxia and tissue ischemia.In this study, we investigated on admission serum UA levels as a potential predictor of in-hospital and 20 months follow up period mortality and morbidity in patients with MI.The aim of this cross-sectional study was to evaluate the effect of on admission serum UA on the short term and mid-term mortality and morbidity of patients admitted with STEMI.We studied 608 patients admitted from December 21, 2012 till February 19, 2014 at Shahid Madani hospital, Tabriz, Iran. Patients were divided into two groups according to their serum UA level.10Patients with STEMI were enrolled in this study. Acute MI was defined as presence of ST-segment elevation consistent with the MI at least 2 mm in adjacent chest leads and/or ST-segment elevation at least 1 mm in 2 or more standard leads or new left bundle branch block, concordant with positive cardiac necrosis markers.11 Having fasting blood glucose level higher than 126 mg/dL or having a random blood glucose test level of 200 mg/dL or higher as well as those taking medications for hyperglycemia were defined as diabetes.12 Hyperlipidemia was defined as cholesterol level ≥200 mg/dl and triglyceride ≥250 mg/dl.13Hypertension was interpreted as blood pressure ≥140/90 or taking anti-hypertensive medications.Patients with liver disease, progressive kidney disorders (creatinine >1.8), gout, alcoholism or taking antihyperuricemic drugs were excluded. Patients with previous history of diuretic and losartan use, also patients with previous history of MI, were excluded.14 Transthoracic echocardiography was used to evaluate LVEF.Blood samples were taken as soon as possible after admission. Laboratory data was gathered and UA level measurement was performed according to the standard procedures of our laboratory at Madani heart hospital, Tabriz, Iran. High UA level was defined as more than 8 mg/dL in men and more than 7.5 mg/dL in women.t test was used to compare continuous variables and Chi-square test was used to compare qualitative variables. UA and lipid profiles relationships were calculated with Pearson correlation coefficients. P value <0.05 was considered significant.SPSS version 17.0 was used for statistical analysis. Continuous data were expressed as mean ± SD/SE. The 7 the statistical power of this study calculated to be 90%.With a sample size of 608 patients and based on previous studiesP = 0.001). P = 0.02), but Increased blood pressure was seen more frequently in high UA group (P = 0.009), and diabetes mellitus and cholesterol level were not different in two groups .In this cross-sectional study, we enrolled 608 patients with definite diagnosis of STEMI. Male to female ratio was 2.53, the mean age of patients was 62.6 ± 13.4, and patients were divided into two groups according to their serum UA level. P = 0.001) and higher creatinine level (P = 0.001). P = 0.001).In comparison with normal UA group, high UA group had significantly higher cTnI , but no association was observed between the serum UA level and ventricular tachycardia. Short term and midterm mortality (average follow up was 20 months) were not different between two groups . We found that serum UA level was associated with higher AF occurrence  was higher than that of our study (62 years old).15 In addition, previous studies revealed that high serum UA level in patients with MI accompanied poor clinical prognosis such as heart failure, low LVEF, and high LV end diastolic diameter (LVEDD).17Some studies have shown that in patients with MI, high serum UA level can increase the mortality rate.19 In this study, we showed the association between high UA level and serum Cr and triglyceride levels. Tuomilehto et al and Nagahama et al reported similar results.21 Nagahama et al showed positive correlation between increased serum UA level and obesity, increased blood pressure and dyslipidemia.In epidemiologic studies, high UA level has been established as a risk factor for the cardiovascular disease.7 In this study, no correlation was found between high serum UA level and hypercholesterolemia.Also Chen et al showed a significant association between UA level and dyslipidemia.23 Huang et al showed that in patients with AF who had not obstructive CAD, coronary blood flow had independent association with serum UA level.We found that higher UA level had association with higher AF occurrence. Similarly Huang et al and Tamariz et al reported higher UA level in patients with AF rhythm.24 Maisel et al showed that incidence of AF was increased significantly in patients with more severe heart failure.25 However, we did not evaluate the frequency of arrhythmias using Holter monitoring and our arrhythmia evaluation was focused on sustaining the symptomatic arrhythmias.Pozzoli et al showed that the onset of AF in patients with heart failure and sinus rhythm was associated with significant worsening in patients’ NYHA functional class.26 In patients who develop heart failure after MI, tissue hypo perfusion and hypoxia lead to the activation of xanthine oxidase and other oxidative products.28 Viña et al reported that the xanthine oxidase is responsible for free radical production. They showed that tissue damage can be caused by the xanthine oxidase activity during heavy exercise. In their study allopurinol could reduce the oxidation of glutathione.29 Moreover, free radicals might be responsible for the inefficacy of re-perfusion therapy. Serum UA level can predict coronary blood flow in patients with acute STEMI treated with primary PCI.30 Okada et al reported that IL-1 induced free radicals stimulate PAI-1 in rats. In addition, they suggested that inhibition of fibrinolysis may be responsible for the serious cardiac dysfunction after the ischemia.31 However, the exact effect of fibrinolysis on the free radicals production is not clearly understood.In this study a significant correlation was found among high serum UA level, low LVEF, and higher Killip class, and Patients with high serum UA level were older, and had higher creatinine. These factors may be responsible for lower percutaneous coronary intervention (PCI) therapy and subsequently higher Killip class in these patients. There are other hypotheses about how high UA level is associated with low LVEF. One of these mechanisms focuses on the impact of UA on the efficacy of thrombolysis. Purine catabolism produces UA by the act of xanthine oxidase enzyme. UA is released in hypoxic conditions.32Kaya et al showed that elevated serum UA level on admission was an independent factor of short term and long term adverse events in patients with STEMI who were treated with primary percutaneous angiography.Evaluating the efficacy of some medications to reduce oxidative stress  after thrombolytic therapy may be helpful and we need large prospective studies to evaluate the effect of serum UA lowering agents in reducing adverse events in patients who develop acute STEMI.These findings suggest that, serum UA level can be used as a prognostic factor in MI patients.This study had some limitations. We did not have 24-hour electrocardiogram (ECG) monitoring and our data only consisted of sustained AF and VT. Because of small number of patients who developed arrhythmia, more investigations with larger sample size can help in better evaluation of association of AF and VT and serum UA level. Finally because IHD is a chronic disease, long term follow-up is needed for better evaluation of long term effects of UA on the cardiovascular system.In the current study, high serum UA level in patients with acute MI was not associated with higher in-hospital or midterm mortality rate. However, high serum UA level was associated with lower LVEF, higher Killip class and AF, elevated cTnI, creatinine, and triglyceride level.We would like to thank all the patients, nurses, and doctors of Shahid Madani heart center.This study approved by the local ethical committee.None to be declared.


While mental health professionals have focused on concerns about whether antidepressants work on a neurochemical level it is important to understand the meaning this medication holds in the lives of people who use it. This study explores diversity in the experience of antidepressant users.One thousand seven hundred forty-seven New Zealand antidepressant users responded to an open-ended question about their experience of antidepressants. This was analysed using content and thematic analysis.There was considerable diversity in participants’ responses including positive (54 %), negative (16 %) and mixed (28 %) experiences with antidepressants. Those with positive experiences saw antidepressants as a necessary treatment for a ‘disease’, a life saver, a way of meeting social obligations, dealing with difficult circumstances or a stepping stone to further help. Negative themes described antidepressants as being ineffective, having unbearable side effects, undermining emotional authenticity, masking real problems and reducing the experience of control. Mixed experience themes showed how participants weighed up the unpleasant side effects against the benefits, felt calmer but less like themselves, struggled to find the one or dosage and felt stuck with continuing on antidepressants when they wished to stop.Mental health professions need to recognize that antidepressants are not a ‘one size fits all’ solution. Antidepressants are being prescribed at high levels across the developed world , 2. In NResearch suggests a range of factors predict different experiences of antidepressants, including demographic factors, psychosocial factors, people’s belief about depression, and their relationship with their prescriber . Some usAn understanding of users’ own experiences of antidepressant use is increasingly important in the context of growing doubts about the effectiveness of antidepressant treatment , 26, conThis article analyses the responses of a large sample of antidepressant users to an open-ended survey question on the impact that antidepressants had on participants’ lives. Our analysis aims to explore the potential diversity of experiences with antidepressants and the meanings attributed to them.This article draws from responses to one open question in an anonymous online survey exploring the experience of adult New Zealanders prescribed antidepressants. The question asked participants to complete the phrase: “In my life antidepressants have been…” The study was approved by the University of Auckland, Human Participants Ethics Committee and participants gave informed consent for their participation.Participants were informed of an online survey via widespread media advertising. The criteria for participation included having been prescribed antidepressants in the last five years, living in New Zealand and being 18 years of age or over. The survey was available online from March 2012 until January 2013. There were 1,829 completed surveys for analysis. The majority of the sample was women (77 %). The modal age group was 36-45 (24.2 %); 16.3 % were 18 to 25, and 15.9 % were 56 or older. Population figures suggest that 13.7 % of the New Zealand population is 65 or older  while onNearly all participants (97 %) had taken the antidepressants when prescribed them, and 69 % were still taking antidepressants. Just over half (51.7 %) had taken them for more than three years, and 7.8 % for less than three months. Of the 1,715 (93.8 %) who answered a question about which antidepressant they had been prescribed, the most common was Fluoxetine (22.4 %), followed by Citalopram (20.3 %), Paroxetine (8.7 %), Tricyclics (4.5 %) and Venlafaxine (2.2 %). Thirty nine percent reported that they had been prescribed multiple antidepressants.The question analysed in this article was only completed by those who reported that they had actually taken antidepressants following a prescription .Participants’ responses, ranged from one word answers through to about 400 words. In order to analyse this large number of responses an initial content analysis was used to generate a quantitative description of the data and provide an overarching framework for a more detailed analysis . Three pThematic analytic methods were used to identify themes within each of the three content categories. Braun & Clarke’s method provided the framework for this analysis, which involved initially identifying and coding key ideas . This prThe content analysis showed that 54 % (939) of participants gave a positive account of antidepressants in their lives. Sixteen percent (273) of participants reported predominantly negative experiences and 28 % (489) described ‘mixed’ experiences. Two percent of responses did not fall into any of these categories (e.g. an elaboration of symptoms of depression).In keeping with neurochemical explanations of depression many participants described antidepressants as a necessary treatment for a ‘mental illness’ often referring to the ‘serotonin deficiency hypothesis’ which sees depression as a result of a chemical imbalance, as the following participant explained: [Its] just like diabetes – a chemical shortage…I need serotonin uptake inhibitors – simple!” These participants saw antidepressants as a necessary and ongoing treatment for an underlying disease: “I would hope that one day I could stop taking them but realize that for me it is the same as taking heart pill for someone else.”This illness framework sometimes involved resignation that the participant needed to remain on antidepressants indefinitely, as one participant explained: “My GP said that if I had diabetes I would need to take insulin forever, so not to worry that I appear to need to continue to take anti-depressants forever.”While some participants described antidepressants as a rational treatment for a disease, other responses conveyed a stronger emotional investment in antidepressant use. A number simply described them as ‘a life-saver’ conveying a sense of the distress which had led them to take antidepressants and the relief they experienced once on antidepressants: “I can still remember the desperation and pain and if it meant taking them forever I would not hesitate.” A number of people elaborated the idea of antidepressants as a life-saver in more literal form, implying that medication had prevented them from committing suicide. As one participant put it, ‘I truly feel that I would not be alive if I had not taken them”.A third theme within the positive responses characterized antidepressant use as enabling ‘normal’ social functioning. [Antidepressants are] the sole reason I can now function as normally as possible as a human being and a participating member of my family and community.” Part of this seemed to be that antidepressants allowed them to better fulfill the demands of their social roles. As one participant put it: “[they have been] very helpful, they have allowed me to be a better parent than I would have otherwise been, I believe.”A fourth theme was somewhat different, describing antidepressants as a temporary way of dealing with challenging circumstances – including interpersonal and social problems. Participants alluded to a range of difficult circumstances as the following response suggestions: “[Antidepressants are] helpful in enabling me to manage the stresses of job loss and unemployment. I feel that I can cope better with job interviews on them.”I have had such good therapy that I have been able to address the wider issues that had contributed to my mental state. …Without the medication though, I would never have had the ability to do this.In some cases, antidepressants were seen not so much as a solution on their own but as a ‘stepping stone’ to some other kind of strategy or support as the following response suggests:For participants who understood antidepressants this way, the medication was generally seen as a temporary solution with therapy providing the more lasting benefit.For a number of participants negative experiences of antidepressants were underpinned by a belief that antidepressants were simply ineffective: “They were a waste of time and did not help me”. Some actively contested the idea that medication could be more helpful than other self-directed strategies of coping: “I get more benefit from mild to moderate exercise, or energy drinks, or spending quality time with friends.” Other participants wrote that they had initially had expectations that antidepressants would help them and had become increasingly disillusioned with their experience, as the following participant describes: “[They were] greatly disappointing. I wish I had never tried them, because before I tried them at least there was hope that something could have helped.”Each one has had a worse effect than the previous…. I can’t remember them all. It started with memory loss then progressed to me becoming borderline catatonic staring at the wall for hours unable to stand up. Within a few weeks and genuinely terrified. It was a relief to go back to the misery of depression after these experiences.The unpleasant side effects of antidepressants formed a second dominant theme in the negative category of responses with a number of participants explaining that they had struggled with the various side effects of different medicines over what appeared to be a lengthy period of time:For some participants the journey to find an antidepressant that did not produce adverse effects had become the primary focus of their lives: “[It’s been] a never ending struggle to find the right ones that do not produce adverse health problems.”Another of the prevalent theme amongst the negative experience responses related to concerns with emotional numbing and the loss of emotional authenticity. A number of participants wrote of feeling “like a zombie.” One participant elaborated on how he understood antidepressants to ‘work’: “They don’t make the problems go away. They just make me numb enough to not give a shit.” Another explained how the emotional numbness impacted on her life more generally: “By taking the medication I felt alienated from others almost as though I was walking around like a zombie in a kind of bubble.”A related theme suggested that antidepressant use was felt to invalidate the genuine suffering participants had experienced. As the following participant put it: “In my life antidepressants have been prescribed to me to cover up what was wrong, and to me were a fake fix.” Some people also wrote about how antidepressants had led them to tolerate circumstances which they would have done better to address directly: “I believe that I stayed in a relationship that was unhealthy for me, because the antidepressants made me tolerate treatment that was unacceptable.”[It’s] like smoking. When you smoke you know it’s bad for you, but you also feel momentary relief and therefore can’t (or don’t want to stop) because you miss that feeling of being slightly more capable to handle situations.A final negative theme captured the way that participants felt that antidepressants had undermined their control over their lives. For some taking antidepressants seemed to raise fears of personal weakness. For example, several participants wrote about using antidepressants as a sign of failing to “cope” or as a sign of dependency:I felt bullied into keeping taking them and at times told I would not receive therapeutic treatment if I didn’t take them. There felt like no alternative and I felt very trapped into taking them.A small number of participants linked their lack of control while on antidepressants to the lack of power over treatment options they experienced in relation to their treatment more generally:I know they do me good and I am better on them, but they do make me feel physically sick, and not like myself. I seem to be constantly trying life without them, but always go back to them in the end.Many participants wrote about how using antidepressants entailed a constant struggle to balance perceived benefits of the medication with side effects. For example, one participant described antidepressants as “a necessary evil, with very unfortunate side effects in terms of weight gain and sexual dysfunction which lead to me stopping the treatment despite its benefits for my mood.” Participants wrote particularly about sexual side effects and how they had to weigh up the negative impact of antidepressants on their intimate relationships with the benefits they felt antidepressants provided to these same relationships. While some, like the previous participant, had ended up stopping antidepressants because of side effects, others resigned themselves to living with these:Antidepressants have been a two edged sword. I felt less affected by things that would normally distress me while on anti-depressants… [but] when I came off them, my head felt clear, I felt like I was waking up and that I was in touch with myself again.A second theme illustrated how participants felt grateful that antidepressants took the edge off their distress but also struggled with a sense that they did not feel ‘like themselves’:[Antidepressants were] helpful in making my depression less. However, the effects that they had on me as a person and how I treated others is the main reason I came off them. I am a considerate and selfless person and while on the antidepressants I was the complete opposite.Another participant reflected on a similar dilemma in which she appeared to weigh up the benefits in terms of reduced feelings of depression and changes in the way she related to others:The thing is that I have been on them so long that I have no idea what it would be like not to be on them. I would love to come off them but they have become such a ‘normal’ part of my life since I was approximately 15 years old that I am not sure I would cope without them.A third mixed theme showed how participants actively balanced the concerns about being ‘dependent’ on them with their fears about their depression returning if they stopped taking the medication. One participant explained how she become so used to being on antidepressants and was afraid of how things would be without them:They helped me get back on my feet when I was facing a difficult time. However I was never told when to go off them and …have not heard from the doctor who prescribed them to me in years.Some participants implied that there had been little follow-up or advice on how to come off antidepressants as one participant wrote:The withdrawal effects if I forget to take my pill are severe shakes, suicidal thoughts, a feeling of too much caffeine in my brain, electric shocks, hallucinations, insane mood swings. [I’m] kinda stuck on them now coz I’m too scared to come off it.Participants also described how withdrawal effects made it especially difficult to come of the medication:I have been on MANY different antidepressants. None of them were helpful at all to me until I tried Fluoxetine 4 years ago. My life now is greatly improved by taking this medication and a quality of life has returned.In contrast to responses which emphasized balancing concerns and perceived benefits of antidepressants, some attributed their mixed experience of antidepressants to the variable effects of the different medications they had been prescribed while for others they felt there had been changes in their own response to a single antidepressant over time. A few participants described how antidepressants appeared to have been initially effective but became less so over time in spite of increased dosages, and for others their response varied markedly with the different medications they had tried:I have tried almost all antidepressants available under prescription (including combinations), and most worked to varying amounts to start with, then stopped helping, then the dose was increased, then stopped working/made me worse, then dose increased to the maximum, then stopped working, then I was put on something else. I’ve wondered if I would have been better off never starting taking them at all  of the overall sample. Some experienced little benefit from antidepressants while the side effects, particularly more subtle psychological effects such as feeling numb or ‘not like themselves,’ seemed a significant issue, which was also found to affect many participants in the analysis of the general survey data . The resOverall, the purely negative responses were far fewer than the ambivalent responses which point to the struggle that a significant proportion of people may experience in choosing to use antidepressants. The search for the right antidepressant at the right dosage may be a long and frustrating journey for some. Experimentation with dosage may also by risky insofar as research suggests that higher dosages do not necessarily produce increased efficacy but may result in greater problems with withdrawal . For useIt is important for mental health professionals to recognize that antidepressants are not a ‘one size fits all’ solution. They need to enter into dialogue with antidepressant users to explore the meaning antidepressants hold in their lives and the extent to which these enable or constrain their ability to make informed choices about their use. In making decisions about whether to take antidepressants, users should not be given misleading information about a known chemical aetiology in depression. They should instead be fully informed about the existing research on the efficacy of antidepressants relative to or in combination with psychosocial treatments . In addiThe findings of this study should be interpreted in the context of its limitations. The volunteer sample might well favor responses from people who have a stronger investment in the issue, such as those who had been on antidepressants for longer. Certainly, more than half the participants in this study had been on antidepressants for more than 3 years. A volunteer sample is also likely to attract those with stronger views about antidepressants. In this study, 84 % of participants in this study answered in the affirmative to a question about whether they felt antidepressants had reduced their depression, a number well above that suggested by efficacy studies , 26. ThiUsing an internet survey might also have skewed the sample towards a more educated and financially well-resourced group of participants and favoured a more youthful sample. The overrepresentation of women (76.6 %) is a less significant bias as they are prescribed antidepressants at much higher rates than men . ConcernThis study was approved by the University of Auckland Human Participants Committee Reference Number 7340. Potential participants were directed to an online information sheet which provided details about the project including its purpose, safeguards for their anonymity and the uses to which the data would be put. They were asked to demonstrate their consent to participate by then clicking through to the survey.Not applicable.Aggregated survey data can be requested directly from the authors.


Rptor (regulatory-associated protein of mTORC1) from the oocytes of primordial follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We provide in vivo evidence that deletion of Rptor in the oocytes of both primordial and further-developed follicles leads to the loss of mTORC1 signaling in oocytes as indicated by loss of phosphorylation of S6K1 and 4e-bp1 at T389 and S65, respectively. However, the follicular development and fertility of mice lacking Rptor in oocytes were not affected. Mechanistically, the loss of mTORC1 signaling in Rptor-deleted mouse oocytes led to the elevation of phosphatidylinositol 3-kinase (PI3K) signaling that maintained normal follicular development and fertility. Therefore, this study shows that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K signaling cascade that maintains normal ovarian follicular development and fertility.The molecular mechanisms underlying reproductive aging and menopausal age in female mammals are poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) is a central controller of cell growth and proliferation. To determine whether mTORC1 signaling in oocytes plays a direct role in physiological follicular development and fertility in female mice, we conditionally deleted the specific and essential mTORC1 component  In mammals, the pool of primordial follicles serves as the source of developing follicles and fertilizable ova for the entire reproductive lifespan of the organism Mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine kinase and a member of the PI3K-related kinase family Rptor gene in mice – which codes for the specific and essential mTORC1 component Raptor – is embryonic lethal in vivo, we generated mice lacking Rptor specifically in the oocytes of both primordial and further-developed follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We found that deletion of Rptor specifically in the oocytes leads to loss of mTORC1 signaling. However, follicular development and fertility in mice lacking Rptor in their oocytes were not affected by the loss of mTORC1 signaling. Interestingly, PI3K signaling was found to be elevated upon the loss of mTORC1 signaling in Rptor-deleted oocytes, and this activity is presumed to maintain the follicular development and fertility in these mice.Conventional knockout of the Rptor gene was deleted specifically in the oocytes of primordial and further-developed follicles (referred to as OoRptor−/− mice). This was achieved by crossing loxP/loxPRptor mice Gdf-9 promoter-mediated Cre recombinase Rptor in oocytes, we performed western blot analysis on oocytes collected from postnatal day (PD)12–14 OoRptor−/− and OoRptor+/+ mice. We found that expression of Raptor protein was completely abolished in growing OoRptor−/− oocytes  in primordial oocytes, which is the upstream negative regulator of PI3K–Akt signaling, causes global activation of all primordial follicles and premature ovarian failure (POF) Pdk1 (3-phosphoinositide-dependent protein kinase-1), which plays a major role in phosphorylating and activating Akt and S6K1 Pten mutant mice (OoPten−/−) is not completely prevented by treatment with rapamycin in vivoin vitroin vivo results demonstrate that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K–Akt signaling cascade and that this is required to maintain normal ovarian follicular development and fertility.In our earlier study, we showed that constitutively enhanced oocyte PI3K–Akt signaling by loss of Tsc1 (tuberous sclerosis complex 1 or hamartin) in oocytes (OoTsc1−/−), which is a negative regulator of mTORC1, also leads to premature activation of the entire pool of primordial follicles and subsequent POF due to the enhanced mTORC1 signaling in oocytes. Such over-activation of primordial follicles is rescued when OoTsc1−/− mutant mice are treated with rapamycin in vivoDeletion of Rptor−/− females. Such compensatory activation of PI3K–Akt signaling has been seen in mice with both adipocyte-specific and skeletal muscle-specific ablation of RptorIn this study, compensatory activation of the PI3K–Akt signaling cascade was observed when Raptor was missing from the oocytes, and this activity appears to be essential to maintain normal physiological follicular development and fertility in OoloxP/loxPRptor mice Gdf-9 promoter-mediated Cre recombinase Rptor in their oocytes (OoRptor−/− mice). Control mice that do not carry the Cre transgene are referred to as OoRptor+/+ mice. The mice were housed under controlled environmental conditions with free access to water and food. Illumination was on between 0600 and 1800. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the University of Gothenburg and were carried out in accordance with the approved guidelines.Rabbit monoclonal antibody (EP539Y) to Raptor was purchased from Abcam. Rabbit polyclonal antibodies to phospho-S6K1 (T389), phospho-4E-BP1 (S65), and phospho-Akt (S473) as well as rabbit monoclonal antibodies to S6K1 and 4e-bp1 were obtained from Cell Signaling Technologies . Mouse monoclonal antibody to phospho-Akt (T308) was purchased from BD Bioscience . Mouse monoclonal antibodies to β-actin and paraformaldehyde were purchased from Sigma-Aldrich Sweden AB . Western blots were carried out according to the instructions of the suppliers of the different antibodies and visualized using the ECL Prime western blotting detection system . Paraffin and hematoxylin were purchased from Histolab, Sweden.Ovaries were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The paraffin-embedded ovaries were serially sectioned at 8-µm thickness and rehydrated followed by staining with hematoxylin for morphological observation. Ovarian follicles at different developmental stages were categorized based on the well-accepted standards established by Pedersen and Peters. Ovarian morphology was determined based on images taken with a light microscope (Zeiss Axio Scope A1 upright microscope). One or both ovaries from more than three mice of each genotype were used for each time point.4Cl and 17 mM Tris–HCl (pH 7.2). After several washes, oocytes were collected by centrifugation. They were then lysed in a buffer containing 50 mM Tris–HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 20 mM β-glycerophosphate, 1 mM EDTA, 6 mM EGTA (pH 8.0), 1% NP-40, 1 mM DTT, 5 mM benzamidine, 1 mM PMSF, 250 µM sodium orthovanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 µg/mL pepstatin followed by centrifugation at 14,000 rpm for 20 min at 4°C. The supernatants were collected and protein concentrations were measured using the bicinchoninic acid (BCA) protein assay, and equal amounts of proteins were used for western blot.Mice were sacrificed by decapitation, and the ovaries were dissected free of fat and connective tissue using a dissection microscope. The ovaries were then minced with a pair of dissection scissors before being incubated in 0.05% collagenase in Dulbecco's modified Eagle's medium-F12  supplemented with 4 mg/mL bovine serum albumin (BSA), 100 units/mL penicillin, and 100 µg/mL streptomycin. The solution was mixed with frequent agitation and pipetting. After the tissues had mostly been digested by the collagenase, usually within 45–60 min, EDTA was added to this mixture to a final concentration of 40 mM and the mixture was incubated at 37°C with frequent pipetting for another 15–20 min until clusters of granulosa cells or other cells were completely dispersed. The mixture of cells and oocytes was then washed once and cultured in a 6 cm or 10 cm tissue culture dish with the above-mentioned serum-free DMEM/F12 medium for 12 h to allow the granulosa cells and other ovarian cells to attach to the plastic. The unattached oocytes and red blood cells were then recovered by collection of the supernatant and centrifugation at 1300 rpm for 5 min at room temperature. Red blood cells were subsequently removed using a hypotonic buffer containing 144 mM NH


Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H2O2 levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H2O2 to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H2O2 that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H2O2 with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H2O2 correlates with aging, it may not be causative.Hydrogen peroxide (H 2O2 in vivo ► Overall mitochondrial H2O2 increases with age but can be independent of life span ► Increased physical activity leads to a decrease in mitochondrial H2O2 ► Hypotheses dependent on overall mitochondrial ROS can now be assessed in vivo► A mitochondria-targeted mass spectrometry probe measures mitochondrial H Mitocho in vivo , but the in vivo , but thi in vivo . FurthermΔψ) . Consequently the reaction of MitoB with H2O2 should be faster in the mitochondrial matrix (pH ∼8.0) compared to the cytosol (pH ∼7.2), further enhancing its specificity for mitochondrial H2O2. Peroxynitrite (ONOO-) rapidly converts arylboronates to phenols  are present together. Thus the extent of MitoB conversion to its phenol product, MitoP, in vivo will reflect the mitochondrial matrix H2O2 and ONOO- concentrations.The strategy  is basedΔψm) . BioactiΔψm) . To makeΔψm) A. The arΔψm) , who sho phenols , so Mito2O2 concentration in vivo within flies. We then apply this methodology to demonstrate that although average mitochondrial matrix H2O2 does increase in flies during aging, interventions such as dietary restriction (DR) increase life span without altering mitochondrial H2O2. These findings have significant implications for how ROS may contribute to aging.A crucial aspect of this approach is the use of mass spectrometry to measure the MitoP/MitoB ratio. This allows application to whole organisms by chemical extraction, rather than being limited to cells or tissue surfaces, as with optical approaches. To ensure accurate quantification, it is essential to include deuterated internal standards (ISs) of MitoB and MitoP to correct for variability during extraction and detection. An important additional advantage of using TPP is that its inherent positive charge facilitates sensitive detection by mass spectrometry. Indeed, derivatization with TPP is used to enhance detection sensitivity in mass spectrometry . Finally2O2 gave a single product, identified by reverse phase (RP)-HPLC (m/z = 369.1). Addition of excess H2O2 to MitoB gave a UV absorbance spectrum identical to that of MitoP  compared to that of the cytosol (7.2) -HPLC A and masof MitoP B. The cot 285 nm B, giving7 M−1s−1 ); therefol (7.2) C, consis2O2 over simple alkylperoxides (- (100-250 μM) to MitoB (100 μM) led to the rapid formation of MitoP as assessed by UV spectrophotometry and RP-HPLC (data not shown), consistent with the fast (k ∼106 M−1s−1) reaction of phenylboronates with ONOO-  (mΔψ was abolished by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)  A. The ace (FCCP) A. MitoP e (FCCP) . Mitochoe (FCCP) , giving e (FCCP) .Figure 43H]-methylTPP (∼48%). This residual cell association of methylTPP and MitoB is caused by uptake into the cytosol due to the plasma membrane potential and to nonspecific binding  (means ± ranges). Incubation of mitochondria with MitoB in the presence of catalase followed by measurement of the mitochondrial MitoP/MitoB ratio gave a low value that increased in the presence of menadione (+/H+ ionophore nigericin lowered the MitoP/MitoB ratio by ∼56% (2O2 production (-11%) and MitoB uptake (+15%). Nigericin lowers the matrix pH from ∼8.0 to 7.2 . We conclude that the MitoP/MitoB ratio reports on mitochondrial H2O2 production within cells.To determine whether MitoB was converted to MitoP by Hl matrix . Controlenadione C. The K+ by ∼56% C but had0 to 7.2 ; hence tfor 6 hr D. TPP cafor 6 hr , so we mfor 6 hr D. Oxidatfor 6 hr D. FCCP dfor 6 hr . Paralle2O2 within living organisms, the fruit fly Drosophila melanogaster was used. MitoB was injected into the fly thorax (∼50 nmol MitoB/g wet weight), and no signs of toxicity were found up to 48 hr after injection. Assessment of head, thorax, and abdomen showed that MitoB distributed throughout the fly with most being in the thorax  with those incubated under hyperoxia (40% or 65% O2). Hyperoxia increases mitochondrial H2O2 production because mitochondrial superoxide production is dependent on [O2] , or with the inactive isomer d-NAME , indicating that ONOO- does not contribute to MitoB oxidation in our experiments. Preadministering the redox cycler paraquat to flies before injecting MitoB significantly increased the MitoP/MitoB ratio , middle-aged (28-day-old) and elderly (56-day-old) flies  flies A. There d) flies B.Figure 2O2 observed during aging may contribute to increased mortality, or may simply be an epiphenomenon with no functional impact  spin traps in conjunction with LC-MS/MS  was found between the MitoP/MitoB ratio and the amount of MitoB in the fly (2 ∼0.1). Therefore alterations in mΔψ or MitoB uptake do not distort the ability of the MitoP/MitoB ratio to report on changes in mitochondrial H2O2 in vivo.As the uptake of MitoB into mitochondria is dependent on  the fly A. Simila the fly B also sh2O2 in vivo has proven impossible. Therefore it is significant that the MitoP/MitoB ratio can be used to calculate the average concentration of H2O2 inside mitochondria within living flies. The rationale and equations for deriving mitochondrial [H2O2] are developed in the 2O2] within the fly provided two assumptions are made: that conversion of MitoB to MitoP only occurs to any significant extent due to direct reaction with H2O2 inside mitochondria. This assumption is justified for three reasons: (1) most (∼88%) of the MitoB within the fly is mitochondrial, (2) the several-hundred-fold concentration of MitoB within mitochondria will lead to a similar fold enhancement of its second-order reaction with H2O2, (3) and within the mitochondrial matrix MitoB has an ∼4-fold greater rate of reaction with H2O2 than elsewhere. The second assumption is that MitoP/MitoB equilibrates throughout the fly rapidly compared to the 3-6 hr duration of the experiments. This is reasonable, as conversion of MitoB to MitoP by H2O2 is slow , while the distribution of TPP cations equilibrates over minutes in cells  in hours, the second-order rate constant for the reaction of MitoB with H2O2 within mitochondria , and the ratio of the MitoB concentration within mitochondria to that in the whole fly (γ). The value for γ derived in the Prior to this work, calculation of the absolute concentration of mitochondrial Hin cells  and in min cells . This is2O2H]mito, with parameters derived from experimentally accessible values. Applying 2O2H]mito that are shown in 2O2H]mito in the range 104-250 nM with an average of 177 ± 27 nM  and aging in the two models investigated here is either absent or masked by changes in other processes that also determine aging.The development of this ratiometric mass spectrometric technique to assess mitochondrial He damage . To clars of age . This ise damage . Further doubled , even th doubled . These fan e.g., . Further2O2H]mito. Thus the decreased accumulation of oxidative damage with aging caused by these two interventions is not caused by changing [2O2H]mito. Instead, the changes in accumulation of oxidative damage are likely to be due to changes in protection, damage repair, and turnover, rather than to increases in ROS levels. Thus DR may act by upregulating damage prevention, repair, and turnover processes, while the elevation to damage that occurs during increased physical activity may be a consequence of diverting resources, such as the ATP/ADP ratio, away from these processes. Together these findings point toward a greater role for damage prevention, repair, and turnover compared to ROS levels in the accumulation of oxidative damage during aging.Both DR and preventing physical activity led to a decrease in the accumulation of oxidative damage , despite2O2H]mito caused by physical inactivity is particularly informative, as it is often assumed that mitochondrial ROS production should correlate with oxygen consumption. In fact, studies with isolated mitochondria clearly predict the opposite  and streptomycin (100 μg/ml). Jurkat cells were grown in RPMI medium (Invitrogen) supplemented with 10% fetal calf serum (FCS), and cells were maintained at 0.2-2 × 106 cells/ml. C2C12 cells were maintained at subconfluence in Dulbecco's modified Eagle's medium  supplemented with 10% FCS.Cells were cultured at 37°C under humidified 95% air/5% COwhite Dahomey strain of D. melanogaster (negative for Wolbachia) was used as the wild-type background and maintained as described in the 2, 182 mM KCl, 46 mM NaCl, 10 mM Tris base [pH 7.2 HCl]) supplemented with 1% (v/v) blue food dye (FD&C Blue No.1) to visualize successful MitoB delivery. The injection volume was ∼75 nl per female fly (∼1.5 mg wet weight) and ∼45 nl per male fly (∼0.85 mg wet weight) to achieve a final MitoB dose of 50 pmol/mg. Flies (in batches of ten) were rendered immobile with CO2 and were injected into the thorax (left pteropleurite). This site was selected because the target consists principally of fly muscle, with minimal risk of damaging any internal structures/organs. The injection process was not harmful, as 7-day-old flies mock injected with Ringer's alone showed >99% survival after 10 days . The amount of MitoB injected was not toxic to the flies, with 100% survival after 48 hr .The 15d-MitoB and 15d-MitoP), vortexed (5 s), and pulse centrifuged to collect debris. The supernatant was transferred to a fresh tube and the fly pellet extracted with a further 200 μl 95% ACN/0.1% FA, which was pooled with the first extraction. The supernatants were then centrifuged , filtered through a 0.22 μm PVDF filter  into a fresh eppendorf tube, and dried under vacuum using a Savant SpeedVac (∼1-2 hr). The dried samples were resuspended in 100 μl 20% ACN/0.1% FA by vortexing for 5 min. Finally, the samples were centrifuged , and 80 μl was transferred to an autosampler vial .To extract MitoB and MitoP from flies, groups of ten were homogenized in a 1.5 ml eppendorf tube using a fitted pestle plunger in 200 μl 95% ACN/0.1% FA. The homogenate was spiked with IS  in positive ion mode using a Waters Quattro Ultima triple quadrupole mass spectrometer attached to a binary pump  and an HTC-PAL autosampler  as described in the 0.99 see B and 3C.


Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL) and tropical spastic paraparesis. Type I interferons are key effectors of innate antiviral response and have been tested as anti-HTLV-1 and anti-ATL agents. HTLV-1 oncoprotein Tax is known to suppress innate interferon response by activating SOCS1, an inhibitor of interferon signaling that activates the expression of interferon-stimulated genes. Whether Tax might also suppress interferon production remains to be determined. Here we report on the suppression of type I interferon production by HTLV-1 Tax through interaction with and inhibition of TBK1 kinase. HTLV-1-transformed ATL cells were found to be unable to boost the production of type I interferons in response to Sendai virus infection. This inability was attributed to Tax. Expression of Tax alone sufficiently repressed the induction of interferon production by RIG-I + PACT, TBK1 and IRF3, but not by IRF3-5D, a dominant active phosphomimetic mutant. This suggests that Tax might act at a point prior to IRF3 phosphorylation. Reciprocal co-immunoprecipitation and immunoblotting experiments confirmed the association of Tax with TBK1 kinase that phosphorylates IRF3. In vitro kinase assay indicated an inhibitory effect of Tax on TBK1-mediated phosphorylation of IRF3. Taken together, our findings suggested a new mechanism by HTLV-1 oncoprotein Tax circumvents the production of type I interferons in infected cells. .


The polymorphisms of IL28B have been described as important in the pathogenesis of infections caused by some viruses. The aim of this research was to evaluate whether IL28B gene polymorphisms (SNP rs8099917 and SNP rs12979860) are associated with HAM/TSP.The study included 229 subjects, classified according to their neurological status in two groups: Group I (136 asymptomatic HTLV-1 carriers) and Group II (93 HAM/TSP patients). The proviral loads were quantified, and the rs8099917 and rs12979860 SNPs in the region of IL28B-gene were analyzed by StepOnePlus Real-time PCR System.A multivariate model analysis, including gender, age, and HTLV-1 DNA proviral load, showed that IL28B polymorphisms were independently associated with HAM/TSP outcome in rs12979860 genotype CT  and in rs8099917 genotype GG .Subjects with SNP rs8099917 genotype GG and rs12979618 genotype CT may present a distinct immune response against HTLV-1 infection. So, it seems reasonable to suggest that a search for IL28B polymorphisms should be performed for all HTLV-1-infected subjects in order to monitor their risk for disease development; however, since this is the first description of such finding in the literature, we should first replicate this study with more HTLV-1-infected persons to strengthen the evidence already provided by our results. New evidence has shown that the pathogenic mechanism of disease-associated HTLV-1 infection is an impairment of the immunity. More recently, it has been demonstrated that IL28B polymorphisms are more likely to occur among HTLV-1 infected subjects and are associated with higher proviral loads in HTLV-1 carriers. Based on anti-HCV properties exhibited by IL28B, we examined the possibility of an association between IL28B polymorphisms (rs8099917 and rs12979860 SNPs) and HAM/TSP occurrence in a large cohort of HTLV-1-infected subjects in Sao Paulo city, Brazil. This study included 229 HTLV-1-infected subjects classified according to their neurological status in two groups (asymptomatic vs HAM/TSP cases), and observed that persons with SNP rs8099917 genotype GG and rs12979860 genotype CT may present a distinct immune response against HTLV-1 infection. Thus, it seems reasonable to suggest that a search for IL28B polymorphisms should be performed for all HTLV-1-infected subjects in order to monitor their risk for HAM/TSP development. Human T-cell lymphotropic Virus Type 1 (HTLV-1) is a retrovirus etiologically linked to adult T-cell leukemia/lymphoma New evidence has shown that the pathogenic mechanism of disease-associated HTLV-1 infection is an impairment of the immunity IL28B is located on chromosome 19, using a cytokine receptor that is specific to the IL10Rβ and IL28Rα, which is expressed in macrophages, dendritic cells and hepatocytes. The signaling receptors for interferons are the result of phosphorylation of JAK1 and Tyk2 kinases, which actively transcribe the factor containing STAT1, STAT2 and interferon regulatory factor 9. Those groups of interferons up regulate viral infections by other interferons, leading to an immune response to HCV The HTLV outpatient clinic from Institute of Infectious Diseases “Emilio Ribas” (IIER) has been following HTLV-infected patients for 15 years, 229 of whom were studied in this project. For the purpose of this study, only patients who are more than 18 years old and were in active follow-up during the period from June 2011 to May 2012 were invited to participate.6 cells and then stored at −80°C. DNA was extracted using a commercial kit  according to manufacturer's instructions. After this procedure the DNA was stored at −80°C for later analysis.The Ethical Board of the IIER approved the protocol (Number 13/2011), and an informed signed consent was obtained from all participants prior to study inclusion. The subjects were classified according to their neurological status in two groups: Group I (136 asymptomatic HTLV-1 carriers) and Group II (93 HAM/TSP patients). Blood samples were collected in acid-citrate-dextrose solution, and PBMC separated by Ficoll density gradient centrifugation . Cells were washed with saline solution; cell number was adjusted to 2×10pol gene: SK110 , and SK111 . The internal HTLV-1 TaqMan probe (5'-CTTTACTGACAAACCCGACCTACCCATGGA-3') was selected using the Oligo  and Primer Express  software programs and checked through a search of GenBank. The probe was located between positions 4829 and 4858 of the HTLV-1 genome and carried a 5' reporter dye FAM (6-carboxy fluorescein) and a 3' quencher dye TAMRA (6-carboxy tetramethylrhodamine). For quantification of the human albumin gene, the primers Alb-S (5'-GCTGTCATCTCTTGTGGGCTGT-3') and Alb-AS (5'-AAACTCATGGGAGCTGCTGGTT-3')and the albumin TaqMan probe (5'-FAMCCTGTCATGCCCACACAAATCTCTCCTAMRA-3') were used as described previously 4 PBMCs, as described elsewhere 4 PBMCs of PVL was the value used as a cut off to discriminate from HAM/TSP subjects.The HTLV-1 proviral load was quantified by real-time PCR, using primers and probes targeting the The rs8099917 and rs12979860 SNPs in the region of IL28B-gene were analyzed by StepOnePlus Real-time PCR System  with the help of a Custom TaqMan SNP Genotyping Assay developed together with Applied Biosystems Stata: Release10. Statistical Software. College Station, TX). The x2 G test for “Goodness of Fit” was used to verify whether the proportions of the polymorphisms were unequally distributed in Hardy-Weinberg equilibrium (HWE)Statistical analysis was conducted using Student's t-test for parametric data, and the chi-square test for proportions. Possible differences in patient characteristics or laboratory values among the groups were evaluated with two-way Mann-Whitney's test and Kruskal-Wallis test. Group analysis was done using Anova test . Bivariate logistic analysis was performed to identify independent variables associated with HAM/TSP. Variables associated with the outcome at a significance level of p<0.20(HAM/TSP) in the bivariate analysis were included in a multivariate logistic model; the only exception to this procedure was the inclusion of the variable gender, which was included regardless of its statistical significance in the bivariate model. Logistic analysis was performed with the aid of Stata 10 software . IL28B genotype distribution at the SNP rs8099917 was: TT , GT  and GG , four patients had no DNA enough to carried out the tests  were female . Sexual he tests . TT genohe tests .2 test for “Goodness of Fit” were x2 22.52, p<0.0001 for SNP rs12979860, and x2 7.63, p = 0.005 for SNP rs8099917, revealing that deviation existed in the Hardy Weinberg equilibrium.A multivariate model analysis, including gender, age, and HTLV-1 DNA proviral load, showed that IL28B polymorphisms were independently associated with HAM/TSP outcome in rs12979860 genotype CT  and in rs8099917 genotype GG . In this model, age was also significantly associated with the outcome when included as a continuous variable . ResultsThe neuropathology of HAM/TSP provides evidence that immune mediated mechanisms may play a significant role in the pathogenesis of the disease The majority of the studies, so far, emphasized the polymorphism SNP rs12979860. However, Soriano's group showed higher proviral loads among HAM/TSP patients, although no association between HAM/TSP and IL28B has been found Thus, for the first time, this polymorphism was associated with clinical outcome, as described in our study. In fact, PVL is important in HAM/TSP patients since they may have up to 5–6 times more HTLV-1 proviral DNA in PBMC when compared to asymptomatic carriers, what may be related to many of the immunologic responses discussed above Similarly, among HCV chronic patients, those who carried the G risk allele at rs8099917 had lower PBMC mRNA expression of IL28B Finally, a tentative explanation: persons with SNP rs8099917 genotype GG may present a distinct immune response against HTLV-1 infection. However, since this is the first description of this finding in the literature, we should first replicate this study with more HTLV-1-infected persons to strengthen the evidence already provided by our results. It is likely that IL28B may have some importance for protection against disease progression Checklist S1STROBE Checklist.(DOCX)Click here for additional data file.


At each count, we noted the number of bird feeders, the number of bird feeders with food, the type of feeders, additional food supplies potentially available for birds  and finally the birds themselves. In winter, urban and rural areas differ in the availability of food offered intentionally and unintentionally to birds by humans. Both types of food availability are higher in urban areas. Our findings suggest that different types of bird feeder support only those species specialized for that particular food type and this relationship is similar in urban and rural areas.Bird feeding is one of the most widespread direct interactions between man and nature, and this has important social and environmental consequences. However, this activity can differ between rural and urban habitats, due to inter alia habitat structure, human behaviour and the composition of wintering bird communities. We counted birds in 156 squares (0.25 kmThe online version of this article (doi:10.1007/s11356-015-4723-0) contains supplementary material, which is available to authorized users. Urbanization is increasing across the globe and it is recognized as a major factor affecting species, populations and assemblages  or in litter bins allowing interspecific competition to play a major role in shaping bird communities at feeding areas. These different forms of supplementary feeding and their effect on wintering birds in different environments have not been previously investigated  across Poland using the same methods  in each of the 26 urban areas in both December 2012 and January 2013. The same numbers of matched rural squares were recorded in the same period, to give a total of 156 squares in each month. Birds were observed during favourable weather conditions  between 8:00 and 14:00 hours. Single observers walked through each square (60–90 min per square) to find all bird feeders, as well as all other potential food sources provided by humans. In each square, we noted the number of bird feeders both with and without food, the type of feeder (details below), additional food supplies potentially available to birds  and finally the birds themselves. To avoid disturbance, birds at feeders and those resting in the vicinity were counted from a distance. For each bird feeder, in both urban and rural squares, a control count in a similar habitat (but without available bird feeders) was also taken at a distance of 100–250 m from the feeder. At all points, birds were counted for a fixed time period of 5 min. This relatively short time was sufficient for the purposes of this study, since wintering birds exhibit a clumped distribution (i.e. with limited movements in order to save energy), bird detection was favoured by the transparency of winter habitats and by the fact that the birds wintering in settlements are accustomed to human presence. The short count time was balanced by the final large sample size (n = 1067) which was necessary to reliably compare bird occupancy at various types of feeders, control points and in two habitats .Bird feeders were recorded by direct observation, located by following bird behaviour, as well as occasionally asking local people. They were counted within three 0.25-kmBird feeders were divided into five categories: 1) typical bird table feeders with a roof providing different types of food, mainly seeds; (2) automatic, mainly bottle-type feeder providing mixed seeds; (3) waste human food, such as bread and boiled vegetables provided usually on the ground; (4) seeds, i.e. mainly wheat and sunflower, placed on the ground or balcony; and (5) pig fat and/or skin sometimes mixed with some seeds and prepared as a block or ball  × 2 habitat type combinations × 17 species) was undertaken in the CANOCO package. A principal components analysis (PCA) was carried out due to a short gradient length of 1.9. This analysis was prepared for the 17 species with more than 100 individuals observed during the study.χ2 = 23.24, df = 4, P < 0.001). In particular, urban-rural differences in feeder types 2 (automatic seed feeder), 3 (waste food) and 5  contributed to the large chi-squared value, while there was little difference in other feeder types  were recorded from 51 species (44 and 45 at bird feeders and control points respectively) Table . The tenP < 0.05. A biplot of the PCA is shown in Fig. Birds were associated with particular types of bird feeder and control points, and the first two axes of the PCA explained 60.4 % of their variability , and both ordination axes were statistically significant at Bombycilla garrulus was the most numerous species associated with the urban habitat, while the characteristic species for rural areas was fieldfare Turdus pilaris.Waxwing Even as the world becomes increasingly urbanized and interconnected, a distinction between urban and rural areas still exists , we showed marked differences between urban and rural areas regarding supplementary feeding of wintering birds. Despite the high scale of bird feeding in both areas, the intensity of feeding and the frequency of five types of feeders were different. Much higher number of feeders and other supplementary food sources were recorded in urban areas confirming greater supplementary food available to birds in the cities. Consequently, twice as many wintering birds were noted in urban compared to rural areas. Moreover, more than twice as many individuals were associated with supplementary feeding locations compared to control locations, both in urban and rural areas. Finally, the composition of bird communities was affected by supplementary feeding. Although species richness was similar in both environments, community composition varied according to the type of feeders; for example, larger species  were particularly associated with waste food in the cities and not in the rural areas.In this large-scale study conducted in 26 locations across Poland  and total numbers of birds recorded at rural and urban feeders and controls. Species are arranged in order of descending overall abundance. (RTF 165 kb)


Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5–50 μM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6–9 h) strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10–15 μM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS. Olea europaea), cranberries, apples, pears and prunes, and some medicinal herbs, such as lavender and salvia, all of them containing the natural pentacyclic triterpene ursolic acid (UA) [The consumption of certain fruits and vegetables of the traditional Mediterranean diet has been associated with low incidence of cancer , 2, provcid (UA) , 4. Thiscid (UA) . Severalcid (UA) , increascid (UA) , supprescid (UA) , 9 as wecid (UA) –12. Morecid (UA) , inhibitcid (UA) , 15 havecid (UA) , 15, 16.The intracellular PI3K/AKT pathway is an important cell signalling route for growth factor-induced proliferation and survival of several cancer cells , 18. OncSoft tissue sarcoma (STS) constitute a heterogeneous group of malignant mesenchymal tumors, for which the standard chemotherapy, based on doxorubicin (DXR) alone or in combination with other drugs, does not achieve significant improvements in terms of overall survival . InteresAlthough many studies evaluated the efficacy of UA in a variety of cell and animal models of adenocarcinoma (for a review see ), only l™), leiomyosarcoma SK-UT-1 (HTB-114™) and fibrosarcoma HT-1080 (CCL-121™) cell lines were obtained directly from the American Type Culture Collection . These cell lines were chosen because they show low sensitivity to doxorubicin (DXR) treatment [The human synovial sarcoma SW982  for 24 h. The compound concentration resulting in 50% inhibition of cell viability (IC50) was determined using GraphPad software. To determine whether UA or TCN would increase sensitivity of these cell lines to DXR, cells were treated with varying concentrations of DXR (range 0.1–10 μM) with or without UA (10 or 15 μM) or TCN (30 μM). After 24 h, the viability of the cells was measured using the methylthiazoletetrazolium (MTT) method. The mean percentage of cell survival relative to that of vehicle-treated cells was estimated from data of three individual experiments each performed in triplicate.Synovial sarcoma, leiomyosarcoma SK-UT-1 and fibrosarcoma HT-1080 cells were plated in 96-well plates at a density of 11 x 103 cells cells were suspended in 0.3% agarose in the corresponding complete growth medium with or without UA (5–50 μM), placed on top of the base layer and cultured at 37°C. Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells were fixed and stained with 0.005% crystal violet dye at 4°C overnight, after 21 or 7 days, respectively. Images were taken at 40-fold magnification from four different fields from each well, and colonies larger than 50 μm were counted as positives.A base layer of 0.6% agarose  in complete growth medium was plated in 6-well plates. Then, 20 x 10The apoptotic index and cell cycle analysis were performed on STS cells by flow cytometry as described previously . Cell poPreparation of cell extracts and protein assays were performed as described previously . PrimarySynovial sarcoma cells were treated with vehicle (DMSO) or DXR (1 μM) in the absence or presence of UA (10 μM) for 2 or 24 h. After this time, cells were harvested, washed and resuspended in PBS at 37°C. Fluorescence attributable to intracellular DXR was immediately measured by flow cytometry (Beckman Coulter Epics XL flow cytometer).P < 0.05. All calculations were done by GraphPad Software.The results are expressed as the mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Bonferroni´s multiple comparison test was used for statistical evaluations. Differences were considered statistically significant at 50 values were 9.03 ± 0.04 μM, 15.04 ± 0.02 μM and 13.42 ± 0.01 μM, respectively . At thetal data . Short-ttal data  and by 1tal data . In paratal data . In conttal data . In agretal data . On the Previous work has suggested that inhibition of AKT could be a desirable therapeutic approach for treatment of STS, in particular, synovial sarcoma. On one hand, activation of the AKT pathway and nuclear β-catenin accumulation has been reported in human synovial sarcoma tissues , 24, 32,PI3KCA and PTEN genes , which should lead to a higher basal AKT activation. Probably as a consequence, UA was still able to induce a significant effect on cell viability, but with lower efficacy compared to the wild type cell line SW982 . For exThe cyclin-dependent kinase inhibitor p21 is another essential protein for proliferation, which has been related to cell cycle arrest and apoptosis . AlthougOleanolic and maslinic acid, structural analogues of UA, also induce apoptosis in SW982 and SK-UT-1 sarcoma cells and sensitize them to DXR . HoweverIn summary, our study reveals that UA not only exhibits a considerable apoptotic effect on STS cells, but also sensitizes them to the current standard chemotherapeutic for this kind of sarcoma, which is DXR. The presented results provide molecular evidence for a possible use of UA in the treatment of certain STS tumors, in particular, synovial sarcoma and leiomyosarcoma. UA is a natural compound present in many dietary components, especially of the Mediterranean diet and, hence, its use as nutraceutical adjuvant together with DXR may ameliorate treatment outcome of patients with STS without increasing the dose of the chemotherapeutic drug or its related toxicity.S1 Fig50) was determined using GraphPad software. *IC50 values of UA were taken from 50 value could not be calculated. Nevertheless, according to the achieved data points in our experimental setting the IC50 value must be higher than 10 μM.Synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells were treated with vehicle (DMSO) or doxorubicin (DXR) (range 0.1–10 μM) alone or combined simultaneously with UA (10 μM for SW982 cells or 15 μM for SK-UT-1 cells) for 24 h. Cell viability was assessed as described in Materials and methods. Each value represents mean ± SEM of 3 independent experiments performed in triplicate. The compound concentration resulting in 50% inhibition of cell viability (IC(TIF)Click here for additional data file.


Human T lymphotropic virus type 1 (HTLV-1) causes a range of chronic inflammatory diseases and an aggressive malignancy of T lymphocytes known as adult T-cell leukaemia/lymphoma (ATLL). A cardinal feature of HTLV-1 infection is the presence of expanded clones of HTLV-1-infected T cells, which may persist for decades. A high viral burden  is associated with both the inflammatory and malignant diseases caused by HTLV-1, and it has been believed that the oligoclonal expansion of infected cells predisposes to these diseases. However, it is not understood what regulates the clonality of HTLV-1 in vivo, that is, the number and abundance of HTLV-1-infected T cell clones. We review recent advances in the understanding of HTLV-1 infection and disease that have come from high-throughput quantification and analysis of HTLV-1 clonality in natural infection. The total number of people infected is at least 5 to 10 million, but the true number is very uncertain, owing to incomplete epidemiological studies in the endemic regions Deltaretrovirus genus of the subfamily Orthoretrovirinae, while the other pathogenic human retroviruses HIV-1 and 2 are classified in the subfamily Lentivirinae. Unlike HIV-1 and 2, HTLV-1 does not cause disease in the majority (over 90%) of infected individuals. Between 1 and 4% of HTLV-1-infected people develop a chronic inflammatory disease, of which the commonest is HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which causes progressive paralysis of the legs HTLV-1 and its congeners HTLV-2, 3 and 4 The history 2+ CD8−; about 4% of cases are CD4− CD8+, and a similar proportion CD4+ CD8+ or CD4− CD8−ATLL is a malignancy of mature, post-thymic T lymphocytes 9 cells L−1), but the cell count rises more slowly, and the patient can remain stable with minor or absent symptoms for months or even years. A proportion of cases (∼20%) present as a lymphoma, with a normal circulating lymphocyte count. This diagnostic classification remains useful for purposes of standardizing clinical trials, comparing disease and treatment outcomes between centres, choosing appropriate therapy and for assessing the prognosis. However, the classification does not reflect the continuum of presentation in the clinic. For example, a purely cutaneous form of ATL lymphoma is recognized, which occurs without leukaemic or nodal disease, and which carries a substantially better prognosis than nodal lymphomas.ATLL was classified into 4 clinical subtypes by Shimoyama et al. 3ATLL carries a poor prognosis because of intrinsic chemotherapy resistance and severe immunosuppression. Despite advances in medical management and supportive care, chemotherapy trials report a median survival of the aggressive subtypes between 7 and 13 months 4gag, pol and env gene products found in other exogenous replication-competent retroviruses, HTLV-1 encodes at least 7 regulatory gene products which control the proviral transcription, mRNA splicing and transport, and the expression of certain host genes. The functions of these regulatory genes of HTLV-1 have been reviewed elsewhere tax and HBZ, appear to play a particularly important role in regulating the expression of viral and host genes and the activation and proliferation of the host cell The genome of HTLV-1 and the major transcripts are shown in HTLV-1 Tax protein has a remarkable range of actions on the host cell, promoting DNA replication and cell-cycle progression, structural damage to the host cell DNA, inhibition of DNA repair and cell-cycle and DNA damage checkpoints, and centrosome over-duplication. Understandably, Tax has therefore been believed to be necessary and sufficient to cause ATLL. Tax is indeed sufficient to immortalize rat fibroblasts in culture, and Tax-transgenic mice develop a variety of tumours + FoxP3+ GITR+, etc.) of the Tax-expressing cell. The loss of Tax results from one of 3 mechanisms: deletion or methylation of the 5′ LTR, or mutation of the provirus A second paradox concerning the putative oncogenic role of Tax is the fact that some 60% of ATLL clones do not express Tax, although the transformed cell typically retains the phenotype  T cells, and 5% in CD8+ T cells HTLV-1 can infect virtually all nucleated mammalian cells in vitro 5.1+ T cells, the main host cell. Within each host the proviral load remains stable over time Early studies found no systematic association between HTLV-1 genotype and disease manifestation Like other exogenous, replication-competent retroviruses, HTLV-1 can propagate both by proliferation of the provirus-carrying cell (“mitotic spread”) and by de novo virion production (“infectious spread”) Oligoclonal expansion of HTLV-1-infected lymphocytes in vivo is frequently easier to detect in patients with HAM/TSP than in asymptomatic HTLV-1 carriers (ACs) 6+ cytotoxic T lymphocyte (CTL) response to HTLV-1. This evidence comes from experiments in host genetics HLA-A*02 was found to give less protection against HAM/TSP in individuals infected with the Cosmopolitan subtype A of HTLV-1 which, as noted above, was associated with a higher prevalence of HAM/TSP in Japan Since HTLV-1 varies little in sequence, and the same viral sequence can occur in asymptomatic HTLV-1 carriers (ACs) and patients with HAM/TSP or ATLL, the observed variation in the outcome of infection among individuals must be chiefly due to variation in the host. There is strong evidence that the principal determinant of an individual's proviral load and risk of HAM/TSP is the HLA Class 1-associated CD8tax and HBZ expression from the provirus plays a central role in the persistence and pathogenesis of HTLV-1 infection The HTLV-1 transactivator protein, Tax, is highly immunodominant in the CTL response to HTLV-1 tax and HBZ gene products promote proliferation of the infected cell, both have been suggested as necessary and sufficient causes of both the oligoclonal T cell proliferation seen in HTLV-1 infection and the pathogenesis of inflammatory and malignant diseases associated with HTLV-1. The potential pathogenic role of these viral gene products must be understood in the context of their normal physiological function in the life history of HTLV-1, since the primary function of these viral genes is not to cause disease in the host but rather to promote survival and propagation of the virus. The central question therefore becomes this: what regulates the expression of the tax and HBZ genes in vivo, and so controls the number, abundance and pathogenicity of HTLV-1-infected T cell clones in vivo?To summarize: since both •How many proviruses are present in each cell?•+ clones are present in a single host?How many distinct HTLV-1•What regulates the abundance of a given clone in vivo?•What regulates expression of the provirus by a given clone?•Does oligoclonal proliferation contribute to the pathogenesis of HTLV-1-induced diseases?To answer this question, we must consider what differs between two clones of T cells naturally infected with HTLV-1. There are three principal attributes that distinguish one infected T cell clone from another: antigen (TCR) specificity, epigenetic modifications, and the genomic site of integration of the HTLV-1 provirus. In addition, as a consequence of the epigenetic modifications, there may be differences among clones in the expression of certain cell surface markers. We have hypothesized that the chief factor that regulates the expression of the HTLV-1 provirus is the integration site of the provirus in the host genome. To test this hypothesis, we recently developed 7The high-throughput integration site protocol 7.1We isolated 28 clones of naturally infected T cells by limiting dilution from the peripheral blood of patients with non-malignant cases of HTLV-1 infection Josefsson et al. 7.2+ clones rises to >60,000.In ATLL, a single HTLV-1-infected clone typically dominates the viral population. In non-malignant cases of HTLV-1 infection, the disproportionate expansion of certain infected T cell clones was first detected by Southern blotting of genomic DNA and by linker-mediated PCR (LM-PCR) + T cell clone live in vivo? It was already clear from the pioneering work of Wattel and colleagues How long does each HTLV-17.3Retroviral integration into the host genome is not random To identify the factors associated with targeting of HTLV-1 integration, we investigated the characteristics of the host genome flanking the integrated provirus, using the high-throughput quantitative protocol 7.4+ clones.The selective oligoclonal expansion of certain HTLV-1-infected T cell clones is a cardinal feature of both non-malignant HTLV-1 infection and, by definition, the malignant disease ATLL. We postulated that the proviral integration site determines the pattern – i.e. the frequency and intensity – of spontaneous proviral expression, which in turn determines the selective expansion of particular HTLV-1Fresh unstimulated PBMCs taken from an HTLV-1-infected person usually do not express detectable levels of HTLV-1 antigens, but strong Tax protein expression becomes detectable after about 6 hours’ incubation in vitro The results The observation that Tax expression is suppressed by the presence upstream of either chromatin remodelling factors or an active host transcriptional start site strongly suggests that the dominant interaction between the flanking host genome and the provirus is transcriptional interference: that is, the inhibition of transcription of the provirus from the 5′ LTR by the presence of an active nearby host promoter upstream of the provirus. It is probable that transcriptional interference contributes to silencing of other integrated proviruses, and it may therefore help to maintain the reservoir of latent HIV-1 7.5It has been widely believed that oligoclonal expansion of HTLV-1-infected T cells is not only responsible for persistence of the infection in vivo but also maintains the high proviral load and predisposes to both inflammatory and malignant diseases associated with HTLV-1. A strong advantage of the recently developed high-throughput proviral sequencing protocol is the ability to quantify accurately the abundance of each HTLV-1-infected T cell clone. This in turn makes it possible to identify the factors associated with the selective expansion of certain clones in vivo. We found that the chief determinants of clonal abundance were the transcriptional orientation of the provirus and its position (upstream or downstream), relative to the nearest host transcriptional start site. Proviruses integrated within a host gene were significantly more frequent in clones of high abundance in vivo than in those with low abundance, but only when integrated in the same transcriptional sense as the host gene.Because of the known mitogenic properties of Tax, we postulated that Tax-expressing clones would reach a higher mean abundance than non-expressing clones in the circulation. But again the results confounded expectation: the frequency of Tax expression was significantly greater in low-abundance clones  [80]. Al7.6Since the proviral load is higher in HAM/TSP patients than in asymptomatic HTLV-1 carriers, and oligoclonal proliferation is frequently detected more easily in samples from patients with HAM/TSP A widely used entropic index, the Shannon index, is of very limited usefulness here because this index is correlated with the sample size, which can be very large in high-throughput sequencing. We therefore defined These observations lead to the general conclusion that oligoclonal proliferation is not a major contributor to the pathogenesis of the associated diseases: clonal expansion is a feature of established ATLL but the expansion per se is not responsible for the malignant transformation. Rather, the proviral load and the risk of inflammatory or malignant disease are determined by the large number of low-abundance clones: these are the clones that frequently express Tax 7.7The understanding of clonality in ATLL is less advanced than in non-malignant HTLV-1 infection, and further work is required. It is widely assumed that ATLL is a monoclonal disease, and indeed in a typical case of acute ATLL a single clone usually dominates. However, there are indications that clonality in ATLL is not always simple. First, there are often many HTLV-1-infected T cell clones underlying the largest, putatively malignant clone We postulate that HTLV-1 constitutes the first ‘hit’ of the 5–8 hits – usually an alteration in a driver gene – that are thought to cause malignant transformation The intense current effort in cancer genomics has led to the identification of a relatively small number of mutations that drive malignant transformation, as distinct from the adventitious and passively propagated “passenger” mutations. These “driver” mutations have recently been classified by Vogelstein et al. In this review we have defined HTLV-1-infected T-cell clones on the basis of their proviral integration site, which indicates a common cell of origin. There is emerging evidence of the importance of subclones in cancer. Recent single-cell sequencing analysis of renal cell carcinomas and myeloproliferative disorders has identified minor subclones carrying rare genetic changes that may contribute to tumour progression. Aggressive ATLL is characterized by rapid relapse following chemotherapy; however, it remains unknown whether such relapse is more commonly due to the emergence of subclones of the same malignant clone that carry additional genetic changes (tumour drivers) or rather to clonal succession None declared.


The human T cell lymphotropic virus type-1 (HTLV-1) infect mainly T cells, dendritic cells and macrophages inducing T cell proliferation and increasing production of chemokines and cytokines by peripheral blood mononuclear cells (PBMC). However, as HTVL-1 may modify the immune response to other infectious agents, the aim of this study was to evaluate the humoral and cellular immune response before and after vaccination with tetanus toxoid (TT). Participants included 14 HTLV-1 carriers and 12 healthy subjects (HS). These individuals were immunized with two doses of tetanus toxoid vaccine. Antibodies to TT were determined by ELISA and the frequency of T cells expressing cytokines as well as the frequency of monocytes expressing co-stimulatory molecules were determined by FACS. The IgG titers anti-TT increased after immunization in both groups (p = 0.001), but HTLV-1 patients had lower levels of IgG anti-TT after immunization when compared with HS (p = 0.007). The frequency of CD4+ T cells expressing IFN-g, TNF and IL-10, after stimulation with TT, was lower in HTLV-1 infected subjects than the HS after immunization (p < 0.05). TNF and IL-12 expression by monocytes after stimulation with TT were higher in the HTLV-1 group than in HS. However there was an impairment in the HLA-DR expression by monocytes from HTLV-1 infected subjects. These results indicate that HTLV-1 infected subjects have a decreasing in humoral response and an impairment in both antigen presenting cells and T cell functions to a biased antigen. Financial Support: INCT-DT, CNPq, NIH R01.


We previously showed that canonical TGFβ signaling is regulated in part by the primary cilium, and that ciliary TGFβ signaling is upregulated in stem cells differentiating into cardiomyocytes . Ciliary


The estrogen receptor and glucocorticoid receptor are members of the nuclear receptor superfamily that can signal using both non-genomic and genomic transcriptional modes. Though genomic modes of signaling have been well characterized and several behaviors attributed to this signaling mechanism, the physiological significance of non-genomic modes of signaling has not been well understood. This has partly been due to the controversy regarding the identity of the membrane ER (mER) or membrane GR (mGR) that may mediate rapid, non-genomic signaling and the downstream signaling cascades that may result as a consequence of steroid ligands binding the mER or the mGR. Both estrogens and glucocorticoids exert a number of actions on the hypothalamus, including feedback. This review focuses on the various candidates for the mER or mGR in the hypothalamus and the contribution of non-genomic signaling to classical hypothalamically driven behaviors and changes in neuronal morphology. It also attempts to categorize some of the possible functions of non-genomic signaling at both the cellular level and at the organismal level that are relevant for behavior, including some behaviors that are regulated by both estrogens and glucocorticoids in a potentially synergistic manner. Lastly, it attempts to show that steroid signaling via non-genomic modes may provide the organism with rapid behavioral responses to stimuli. Nuclear receptor ligands such as estrogen and glucocorticoids signal via both non-genomic and genomic pathways within cells. The genomic or transcriptional pathway is the best elucidated primarily due to the well-characterized nature of the estrogen receptor (ER)α and β and the glucocorticoid receptor (GR), all of which are members of the nuclear receptor superfamily. Once bound to their cognate ligands, these receptors act as ligand-activated transcription factors in the nucleus by binding to specific enhancer elements such as the estrogen response element (ERE)  and glucOn the other hand, non-genomic signaling, first described by Szego and Davis in 1967, as the rapid increase in cAMP in the uterus occurred within 15 min of 17β-estradiol (17β-E) administration to ovariectomized mice . In the Central to this concept of non-genomic signaling that is typically demonstrated by the use of membrane-limited conjugates , is the 3H-labeled 17β-E    showed r(E2-BSA)  have sug(E2-BSA)  that the(E2-BSA) . Most stAs we will focus on outputs dependent on the hypothalamus in this review, we will initially discuss the nuclear classical ERα as a candidate mER because (a) this is the most abundant ER isoform in the medial preoptic area (mPOA), ventromedial hypothalamus (VMH), and arcuate nucleus (ARH) –27 and  and aggrq and ERK signaling. This, in turn, increased pCREB activation within 5 min of 17β-E application   . Though synthase , this mos and Gαq subunits in the CHO cell line ; the corresponding site in the mouse ERα is the C451 . Mutant ell line  -β-arrestin-dependent manner , 17β-E ain vivo, particularly in light of an absence of a specific antibody to the ERβ  channel, in a PLC-, PKC-, and PKA-dependent manner  and MDA-MB-231  breast cancer cell lines  that exists as a complex with heat-shock protein 90 (HSP90), p23, and a tetratricopeptide protein  in the canscript . GR exisanscript . GRβ neianscript  and thisanscript  such as anscript . Similaranscript . Howeveranscript , suggestanscript . Hypothaanscript . Synaptoanscript  and GR ianscript  while inanscript . Functioanscript . The hypanscript . Bath apanscript . The relH neuron . HoweverH neuron  onto theH neuron . Hence, q-coupled novel proteins are the mERs in some tissues, it is also possible that they crosstalk with classical ERα or ERβ, accounting in some studies for the ICI-mediated antagonism of the effect. Though this cooperation between GPR30 and ERα has been shown to have a proliferative effect on ovarian cancer cells  injected into the lumbar muscles labeled the lordosis circuit, in particular, the plexus of oxytocin fibers in the vl-VMH . In the  density  upon EB  density . The pro density .Does non-genomic signaling play a role in the increase in spine density by estrogens or by glucorticoids? The mGluR1a antagonist, LY367385 in the ARH decreased phosphorylated cofilin levels that are induced within an hour of EB injection and that are required for actin reorganization and spinogenesis, suggesting that at least some of the initial aspects of spinogenesis are mediated via non-genomic signaling from the ERα-mGluR1a complex at the membrane . Though Consistent with the idea that 17β-E-mediated spinogenesis in the VMH has a functional consequence, cytochalasin D, an actin polymerization inhibitor, blocked the formation of spines in the ARH and reduced lordosis  though tq-mER may couple to mGluR1a and facilitate lordosis, though the mechanisms by which this occurs are unclear. Apart from these receptors, the localization of the GPR30 in the hypothalamus  and spinal cord motor neurons, resulting in the classical lordosis posture . A seriethalamus  and the thalamus  argues t−/AA)   did not −/AA) , demonst−/AA) , demonst−/AA)  shows th−/AA) , both pr−/AA) .−/AA mouse abolished male sex behavior (In males, 17β-E given intraperitoneally at high concentrations to castrated adult rats increased some aspects of male sex behavior, i.e., genital sniffs and mounting within 15 min, a time frame commensurate with non-genomic actions . Howeverbehavior , it is rbehavior , 138, tebehavior . In the behavior . This apbehavior . This imbehavior , the exabehavior . Stimulibehavior , a nuclebehavior . In thisCastration in male rodents removes both testosterone and estrogens and results in loss of aggression in response to territorial intrusion , 145. ThWhat is the source of 17β-E? In rodents, aromatase is expressed in axon terminals –152 partThough 17β-E can rapidly increase aggression by modulating neurotransmitter release [ and refeGonadal status can modify the actions of glucocorticoids on the hypothalamus or can regulate corticosterone levels itself. For example, gonadal steroids can modify glucocorticoid-mediated negative feedback on CRH. 17β-E chronically administered to gonadectomized and adrenalectomized female rats treated with high doses of corticosterone increased CRH mRNA levels, thus counteracting negative feedback, while DHT treatment to male rats had an opposing effect and caused a further decrease in CRH levels . In femaOne function of non-genomic signaling within a cell could be to provide cells with another pathway by which they can respond differently to the same endogenous ligand. For example, acute signaling by glucocorticoids increases aggression in an ethologically relevant context but chronic signaling by glucocorticoids increases submission . A paralUnderstanding the many facets of non-genomic signaling would also spotlight the role and source of the ligand. The idea that local estrogen synthesis in the brain, i.e., neuroestrogens is important in transducing environmental stimuli to behavior is fairly recent and has mostly been explored in birds , 196. InComplementary studies using deletions of the genes in animals or preferably using site-specific or temporally specific deletion would prove useful to establish specificity. The detailed molecular pathways by which 17β-E regulates spinogenesis or behaviors in the VMH are also not well worked out, though there has been considerable progress in the last decade on the receptors that might be involved. Lastly, understanding non-genomic signaling is possibly a mechanism to convey information about external physiologically relevant stimuli rapidly to the animal to generate both acute and chronic outcomes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although the supernatant p19 levels and proviral loads for all four infected lines were similar, a significant difference in Tax-specific CTL killing was observed. Cells infected with orf-I-knockout virus, p12-G29S or p8-N26 were killed whereas cells infected with WT virus were not killed by Tax-specific CTLs. These results indicate that efficient viral persistence and spread requires expression of both p12 and p8.HTLV-1 orf-I is important for immune evasion, viral replication and persistence. Here, we analyzed proviral sequences isolated from ex vivo PBMC samples of 163 HTLV-1-infected patients . Of the 5000 sequences analyzed, orf-I expression was consistently maintained. While the highest percentage of isolates express both p12 and p8 (69%), the mutants that express mainly p8 (7%) or mainly p12 (24%) were highly correlated with decreased proviral loads. To determine the individual role of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing either p12(G29S) or p8(N26) and established producer cell lines used to infect macaques. Three of four macaques infected with WT-HTLV-1 tested positive by PCR and were seropositive. Only one of the macaques infected with N26-HTLV-1 tested positive by PCR and two macaques were seropositive. None of the 8 animals infected with G29S-HTLV-1 tested PCR positive or seroconverted. These results suggest that expression of both p12 and p8 are critical for viral persistence. Since orf-I plays a role in T-cell activation and recognition, we compared the CTL responses elicited by CD4