The primary treatment of hemorrhagic shock is control of the source of bleeding as soon as possible and fluid replacement. In controlled hemorrhagic shock (CHS) where the source of bleeding has been occluded fluid replacement is aimed toward normalization of hemodynamic parameters. In uncontrolled hemorrhagic shock (UCHS) in which bleeding has temporarily stopped because of hypotension, vasoconstriction, and clot formation, fluid treatment is aimed at restoration of radial pulse, or restoration of sensorium or obtaining a blood pressure of 80 mmHg by aliquots of 250 ml of lactated Ringer's solution (hypotensive resuscitation). When evacuation time is shorter than one hour  immediate evacuation to a surgical facility is indicated after airway and breathing  have been secured ("scoop and run"). Precious time is not wasted by introducing an intravenous line. When expected evacuation time exceeds one hour an intravenous line is introduced and fluid treatment started before evacuation.Crystalloid solutions and blood transfusion are the mainstays of pre-hospital and in-hospital treatment of hemorrhagic shock. In the pre-hospital setting four types of fluid are presently recommended: crystalloid solutions, colloid solutions, hypertonic saline and oxygen-carrying blood substitutes. In unstable or unresponsive hemorrhagic shock surgical treatment is mandatory as soon as possible to control the source of bleeding. Claude Bernard suggested that the organism attempts to maintain constancy of its milieu interie despite external forces that attempt to disrupt it [Hemorrhagic shock is defined as a condition of reduced perfusion of vital organs leading to inadequate delivery of oxygen and nutrients necessary for normal tissue and cellular function. The understanding of the pathophysiology of shock has made significant progress only in the late 19homeostasis to describe the equilibrium maintained in the internal environment, and is credited for the first proposal to cause deliberate hypotension in order to reduce internal hemorrhage in uncontrolled hemorrhage before control of bleeding vessels [Walter B. Cannon introduced the term  vessels .Alfred Blalock proposed in 1934 four categories of shock: hypovolemic, vasogenic (septic), cardiogenic and neurogenic shock [In 1947 Wiggers developed an animal model of graded controlled hemorrhagic shock by uptake of shed blood into a reservoir to maintain a predetermined level of hypotension [This classic model was used by G. Tom Shires in the 1960s and 1970s to demonstrate that a large extracellular fluid (ECF) deficit occurred in prolonged severe hemorrhagic shock which was greater than could be attributed to vascular refill alone . Only thAggressive fluid resuscitation during the Vietnam War with red blood cells, plasma, and crystalloid solutions allowed patients who previously would have succumbed to hemorrhagic shock to survive. Renal failure became a less frequent clinical problem, vital organ function was better sustained, but fulminant pulmonary failure termed "DaNang lung" or "Acute Respiratory Distress Syndrome (ARDS)" appeared as an early cause of death after severe hemorrhage.Additional studies by this group demonstrated that this prolonged period of hemorrhagic hypotension was associated with the development of microvascular injury with marked ECF deficit which could be corrected only by the administration of isotonic crystalloids in volumes 2 to 3 times the estimated blood loss to achieve survival. This was the basis of the current well known dogma "3 to 1 rule" for the treatment of hemorrhagic shock, which was adopted by the ATLS for the treatment of trauma casualties . It was This practice however, has recently been challenged in clinical trials ,8 and exThis fundamental dissimilarity in the hemodynamic response between controlled hemorrhagic shock (CHS) in which the bleeding source has been occluded, and uncontrolled hemorrhagic shock (UCHS) in which bleeding has temporarily stopped because of hypotension, vasoconstriction, and thrombus formation, constitutes the basis for our guidelines of fluid resuscitation in civilian as well as military trauma .In CHS where the external bleeding source has been occluded as well as in UCHS where bleeding has temporarily stopped due to hypotension, when evacuation time is estimated to be shorter than one hour , immediate evacuation to a surgical facility is indicated after the airway and breathing  have been secured ("scoop and run"). Precious time is not wasted by introducing an intravenous line before evacuation, but infusion can be started en route to the medical facility.When evacuation time is expected to be longer than one hour, an intravenous line is introduced and fluid resuscitation is started before evacuation.Fluid resuscitation in CHS is aimed toward normalization of hemodynamic parameters, in contrast to UCHS, where hemostasis cannot be safely achieved, and early rapid evacuation to a surgical facility is considered the most important step of management after the airway and breathing have been secured. If transportation means are readily available fluid treatment can be started during evacuation.According to the guidelines of the Israeli Defense Forces (IDF) fluid resuscitation of CHS is aimed toward normalization of hemodynamic parameters, in contrast to UCHS where the principles of hypotensive resuscitation are operative and treatment is started when one of the three parameters is documented :a. Altered sensoriumb. Radial pulse cannot be palpatedc. Systolic blood pressure dropped below 80 mmHg.Fluid treatment does not include the automatic early administration of 2000 ml of lactated Ringer's solution as recommended by the ATLS guidelines, but repeated aliquots of 250 ml are administered with continuous monitoring, aiming at a systolic blood pressure of 80 mmHg, appearance of a radial pulse, or regained consciousness. In several studies it has been demonstrated that the prognosis of brain injuries is primarily dependent on cerebral perfusion . TherefoAggressive fluid infusion to achieve normal hemodynamic parameters is prohibited in UCHS because it may renew internal bleeding ("pop the clot"). Massive fluid resuscitation is withheld until the time of the surgical intervention. When the expected transportation time to the medical facility exceeds one hour, hemodynamic evaluation is repeated every 15 minutes, and if systolic blood pressure drops below 80 mmHg, radial pulse cannot be palpated or the sensorium deteriorates, aliquots of 250 ml of Ringer's lactate solution are infused in order to maintain this blood pressure.Infusions of crystalloid solutions and blood transfusions are the mainstays for the pre-hospital and in-hospital treatment of severe hemorrhagic shock. Blood is required for repletion of oxygen-carrying capacity, but it is usually not readily available in the prehospital settings because it requires refrigeration and typing. In the pre-hospital setting there are 4 types of fluids that are presently recommended for treatment of hemorrhagic shock:Lactated Ringer's solution is the most widely available and frequently used balanced salt solution for fluid resuscitation in hemorrhagic shock. It is safe and inexpensive, and it equilibrates rapidly throughout the extracellular compartment, restoring the extracellular fluid deficit associated with blood loss. Because of the rapid equilibration of balanced salt solutions into the extracellular space, larger volumes may be required for adequate resuscitation, resulting in decreased intravascular oncotic pressure. Although the use of crystalloids has been routine for resuscitation of patients with acute blood loss, several studies have raised questions regarding the effects of resuscitation regimens on aspects of the immune response to hemorrhagic shock. It was observed by Rhee et al.  that lacThe use of colloid solutions that tend to remain in the intravascular compartment has been advocated for treatment of hemorrhagic shock. Several colloid solutions were studied in clinical practice including human albumin, hydroxyl ethyl starch (HES), and dextran. Because colloid solutions remain briefly in the intravascular compartment, a lower total volume of resuscitative fluid is required to attain hemodynamic stability compared to crystalloid solutions. However, colloid solutions are more expensive, may bind and decrease serum ionized calcium, decrease circulating levels of immunoglobulines, and may further compromise the extracellular fluid volume deficit rather than restoring it. Numerous experimental and clinical studies have compared crystalloid and colloid fluid resuscitation ,23. TherClinical and experimental studies have demonstrated that a small volume of hypertonic saline (5 ml/kg NaCl 7.5%) with or without dextran can be an effective initial resuscitation solution. Hypertonic solutions improve microvascular flow, control intracranial pressure, stabilize arterial pressure and cardiac output with small-volume infusion, with no deleterious effects on immune functions -27. Basehold promise as effective resuscitation fluids that may improve oxygen carrying capacity without problems of storage, compatibility, and disease transmission that are associated with standard blood transfusion. Oxygen-carrying blood substitutes can be generally divided into two types: fluorocarbon-based synthetic oxygen carriers and stroma-free cross linked human or non-human hemoglobin products. The fluorocarbon emulsions are easy to produce, have a long shelf life, and have minimal infectious or immunogenic effects. Potential disadvantages include the requirement of a high FiO2, and rapid plasma clearance. Hemoglobin-based oxygen carriers are notable for high oxygen carrying capacity, an appreciable oncotic effect, and prolonged shelf life. Disadvantages include short plasma half life, potential renal toxicity, hypertensive effects, and the potential of immunogenic effects. Further clinical trials to establish the optimal dosage, efficacy, safety, and the effect on outcome are indicated before oxygen-carrying blood substitutes are implemented in routine clinical practice.Once oxygenation and circulating volume have been restored, re-evaluation of the clinical situation is in order. Vital signs, mental status, urinary output, and capillary refill should be assessed regularly throughout the resuscitation. Initiation of central monitoring may be indicated at this time, if the response to initial resuscitation has been less than expected, or if blood loss is ongoing. Blood should be drawn to assess hematological, coagulation, electrolyte and metabolic status. Electrolyte and metabolic disorders as well as coagulation deficiencies should be corrected. Arterial blood gases should be obtained to determine the adequacy of oxygenation. Management of alternations in oxygenation, ventilation, pH, fluid and electrolyte balance should now be based on clinical evaluation and laboratory measurement. Blood components may also be used at this stage to replace identified deficiencies.Most cases of unresponsive hemorrhagic shock to fluid management in the trauma patient are due to ongoing losses of blood volume or myocardial dysfunction. While initial stabilization is taking place, attention should be directed to prompt arrest of bleeding. Aggressive restoration of normal blood pressure without arrest of internal hemorrhage will enhance further losses of blood volume by increasing flow and impeding coagulation at the site of injury. Mild to moderate hypotension allows for clot formation and slows bleeding from injured blood vessels (hypotensive resuscitation). The hemodynamically unstable injured victim should be brought to surgery as soon as possible and the source of bleeding promptly identified and arrested.


Modern obstetrics is faced with a serious paradox. Obstetric practice is becoming increasingly interventionist based on empirical evidence but without a theoretical basis for such intervention. Whereas obstetric models of perinatal death show that mortality declines exponentially with increasing gestational duration, temporal increases in medically indicated labour induction and cesarean delivery have resulted in rising rates of preterm birth and declining rates of postterm birth. Other problems include a disconnection between patterns of gestational age-specific growth restriction (constant across gestation) and gestational age-specific perinatal mortality  and the paradox of intersecting perinatal mortality curves .The fetuses at risk approach is a causal model that brings coherence to the various perinatal phenomena. Under this formulation, pregnancy complications (such as preeclampsia), labour induction/cesarean delivery, birth, revealed small-for-gestational age and death show coherent patterns of incidence. The fetuses at risk formulation also provides a theoretical justification for medically indicated early delivery, the cornerstone of modern obstetrics. It permits a conceptualization of the number needed to treat  and a calculation of the marginal number needed to treat . Data from the United States showed that between 1995–96 and 1999–2000 rates of labour induction/cesarean delivery increased by 45.1 per 1,000 and perinatal mortality decreased by 0.31 per 1,000 total births among singleton pregnancies at > = 28 weeks of gestation. The marginal number needed to treat was 145 (45.1/0.31), showing that 145 excess labour inductions/cesarean deliveries in 1999–2000 (relative to 1995–96) were responsible for preventing 1 perinatal death among singleton pregnancies at > = 28 weeks gestation.The fetuses at risk approach, with its focus on incidence measures, provides a coherent view of perinatal phenomena. It also provides a theoretical justification for medically indicated early delivery and reconciles the contemporary divide between obstetric theory and obstetric practice. Increases in medically indicated labor induction and cesarean delivery in recent decades have resulted in increases in preterm (< 37 weeks) birth rates, while births at term 37–41 weeks) and postterm (≥ 42 weeks) gestation are also being delivered much earlier than previously  weeks an. HoweverThe cornerstone of modern obstetrics is selective, carefully timed early delivery given fetal compromise . Medically indicated labor induction and cesarean delivery are typically employed when the balance of risks and benefits indicate that birth and supportive neonatal care are preferable to an intrauterine environment that is adversely affecting fetal well-being.Induction of labor to effect early delivery was introduced in the mid-18th century as a management option for contracted pelvis . In the a decline in perinatal mortality [However, traditional epidemiologic and obstetric models of perinatal death do not support this iatrogenic increase in early delivery -14. Suchortality -9,21-23.rd or < 10th percentile of birth weight for gestational age) suggest that a fixed fraction of births (approximately 3% or 10% depending on the cut-off used) are growth restricted at each gestation. Such an implied constancy of the growth restriction rate across gestation is at odds with an exponentially declining rate of gestational age-specific perinatal mortality. Clearly, this incongruence between patterns of in utero growth faltering and death needs to be reconciled, given the known relationship between fetal growth restriction and perinatal death [The paradox of modern obstetrics is also evident in relation to cerebral palsy. Although preterm birth is highly associated with cerebral palsy and deemed to be an important cause of cerebral palsy ,24, the al death ,26.Other problems in the fetal growth literature relate to fetal growth standards. Some fetal growth standards provide unisex reference values -30, sevelower neonatal mortality rate than infants of non-smokers, the reverse is true at higher birth weight. Are the low birth weight or preterm infants of smokers more healthy than the low birth weight or preterm infants of nonsmokers? Addressing the paradox of intersecting perinatal mortality curves is important because the resolution of scientific paradoxes often leads to greater substantive insights. The contemporary appeal of traditional models notwithstanding, intersecting perinatal mortality curves (and the other above-mentioned conundrums) suggest that there may be a more compelling perspective on perinatal events.Perhaps the most intriguing of the paradoxes in the perinatal literature is presented by intersecting birth weight- and gestational age-specific perinatal mortality curves. Birth weight-and gestational age-specific perinatal mortality curves intersect  when conThe conundrums and paradoxes evident in contemporary perinatology are, at least partly, a consequence of the manner in which time related concepts are addressed in traditional models.Two time scales are commonly used in perinatology and these measure the duration of life in utero  and the duration of life after birth . The clinical problems caused by these dual overlapping scales are generally recognized, especially by clinicians in neonatology, who resort to a single scale for expressing age, namely, post-menstrual age or corrected gestational age. Such recognition is also reflected in the evolution of Bronchopulmonary Dysplasia, which was historically defined as a requirement for oxygen at more than 28 days after birth but now refers to a requirement for oxygen or ventilatory support at 36 weeks of post-menstrual age . An impoGestational age is often treated as a determinant in perinatal epidemiologic studies. As a determinant, gestational age at birth  serves as a powerful predictor of death and other adverse perinatal outcomes. However, from an epidemiologic perspective, gestational age is in fact follow up  time and should be treated as such in causal models.The problem inherent in calculating traditional gestational age-specific stillbirth rates  and equating these estimates with gestational age-specific stillbirth risk was first identified over 15 years ago . Yudkin The incidence rate of any pregnancy related event at any gestation is defined as the number of new cases of the event that occur within that gestational week divided by the number of candidates at risk for the event at that gestation. Thus, the incidence of birth Tables , 2 is capattern of growth restriction with increasing gestational duration i.e., to estimate whether it increases, decreases or remains constant. The measure cannot provide the absolute rate of SGA at any gestation since the numerator of the index is dependant on birth . Improvements in technology will permit quantification of the absolute rate (incidence) of growth restriction at each gestation in the future.The incidence of growth restriction is a good example of an index whose estimation presents a challenge. Although routine obstetric practice includes screening for and diagnosis of growth restriction, the technology is insufficiently advanced to permit valid and complete ascertainment of all new cases at each gestation -65. GiveThe fetuses at risk approach for stillbirth is a survival analysis model with censoring of subjects (fetuses) at birth. This gestational age-specific stillbirth calculation provides estimates of the cumulative incidence of fetal death at each week of gestation and approximates the incidence density (hazard) of stillbirth. The extended fetuses at risk model integrates perinatal death and serious neonatal morbidity  into a single framework Figure  since thAs mentioned, the extended fetuses at risk model most deviates from traditional models with respect to events that occur after birth and yet have a prenatal etiology. Under the traditional model of perinatal death, neonatal deaths occur among infants in the first month after birth and the unborn fetus is not a candidate for neonatal death. However from a broad biological, obstetric and ultimately epidemiologic point of view, a fetus at any gestation is at risk of stillbirth and neonatal death at that gestation. If one considers a woman at 28 weeks gestation with severe preeclampsia and fetal compromise, the risk of stillbirth is easy to conceptualize. The risk of neonatal death is substantial as well and can follow either premature labour or medically indicated delivery. The same risks apply in concept to a woman with a healthy pregnancy at 28 weeks gestation, despite the magnitude of the risks being considerably smaller . Thus, aThe extended fetuses at risk formulation of death provides 2 alternative models that treat time per epidemiologic principles . In the first model, namely, the comprehensive model of death, time is measured on the scale of post-menstrual  age with fetuses/infants censored at death . Birth iThe fetuses at risk formulation brings coherence to the study of perinatal phenomena. It shows that the incidence of pregnancy complications such as preeclampsia and chorioamnionitis increases as gestational age advances ,62. ReveThe fetuses at risk formulation faces its most serious challenge from the traditional idea that perinatal mortality declines as gestational age increases. Indeed, this latter inference appears intuitive and is corroborated by the readily apparent relationship between birth weight and perinatal mortality. Despite the socially important prognostic purpose served by the traditional model of perinatal death, it is not appropriate as a causal model. The use of dual overlapping time scales for life in utero  and after birth (chronologic age) and the truncation of the full biologic continuum  is problematic on the level of first principles and also because it is responsible for numerous paradoxes and conundrums . The entAs for explaining the rise in growth restriction and perinatal mortality rates with increasing gestation, one can speculate that the ability of the utero-placental system to support the fetus declines with increasing gestational age. Rising rates of growth restriction  and perinatal death with increasing gestational duration reflect increases in the incidence of pregnancy complications such as preeclampsia and chorioamnionitis ,62 and aThe above-mentioned arguments suggest that the traditional and fetuses at risk models serve vastly different purposes. The distinction between descriptive versus causal models is particularly relevant in this context ; traditiDeveloping an explicit epidemiologic framework for justifying medically indicated early delivery is important in order to avoid conflicts between obstetric theory and practice . Thus, iThe rate of labor induction and/or cesarean delivery increased with increasing gestational age Figure , being lThe number needed to treat (NNT), an index widely used in therapeutics as part of risk-benefit equations, is insufficiently articulated in connection with medically indicated early delivery . This isThe clinical scenarios described below are characterized by varying background rates of perinatal mortality . The first scenario involves an obstetric emergency  where a rapid absolute increase in the (incidence density) rate of perinatal death is anticipated over a short time span (minutes). It is expected that an emergency cesarean delivery carried out within 15–30 minutes will prevent perinatal death in more than half the fetuses . This imVirtually all estimates used in the NNT calculations above are speculative even if they represent more or less reasonable approximations. In fact, most inputs into the NNT calculation cannot be estimated given current standards of care since the decision not to intervene is such situations  would constitute a breach of ethical standards.The marginal NNT, which measures the effect of increases in medically indicated early delivery , is an alternative measure that is directly pertinent to obstetric practice. In this calculation, a temporal increase in medically indicated early delivery is set against the change in perinatal mortality in any particular domain.Table Such marginal NNT calculations are analogous to calculations based on randomized trials which contrast routine induction of labor vs selective induction of labor at or beyond term  or thoseThe proposed framework is based on two important assumptions. First, medically indicated early delivery is considered the final pathway for obstetric intervention. Thus, increases in labor induction and cesarean delivery are credited with preventing perinatal death even though such early delivery was facilitated by improved methods for assessing fetal well-being and supportive neonatal care. Early delivery is thus viewed as a therapeutic package which subsumes antenatal monitoring, diagnosis of fetal well-being, supportive neonatal care and other interventions that permit higher rates of early delivery to rescue compromised fetuses from a hostile intrauterine environment.A second assumption is that temporal increases in labor induction and cesarean delivery rates and declines in perinatal mortality rates reflect true changes in obstetric practice (rather than changes in population characteristics). It is possible that changes in maternal characteristics  may Current limitations of the fetuses at risk approach include an inability to precisely document the incidence of fetal growth restriction. This is because diagnosis of growth faltering in utero, although much facilitated in recent decades through ultrasonographic means, remains inaccurate and essentially unavailable at the population level -65. The The cornerstone of modern obstetrics, namely, early delivery given fetal compromise, cannot be reconciled with traditional models of perinatal mortality which show that perinatal death rates decline exponentially as gestational duration increases. On the other hand, the fetuses at risk approach, which shows that pregnancy complications, revealed SGA and perinatal death rates increase with increasing gestational age, provides a justification for medically indicated early delivery and also resolves several prevailing conundrums in the perinatal field. Although inputs for estimating therapeutic indices related to medically indicated early delivery (such as the NNT) cannot be obtained for ethical reasons, it is possible to retrospectively estimate the marginal NNT associated with medically indicated early delivery. This provides an estimate of the number of additional medically indicated early deliveries that were required to prevent one perinatal death. On a more general level, the traditional model of perinatal death and the fetuses at risk approach are best viewed as serving different purposes; the former is suited for setting prognosis at birth while the latter provides a causal framework and the basis for obstetric intervention.The author(s) declare that they have no competing interests.KSJ proposed the thesis, carried out the analysis and wrote the manuscript. The paper was presented at a plenary session of the 2004 Annual Meeting of the Society for Pediatric and Perinatal Epidemiology, Salt Lake City, Utah.The pre-publication history for this paper can be accessed here:


Dietary supplement use is widespread in developed nations. In particular, patients who utilize mental health services also report frequent consumption of dietary supplements, often in relation to management of adverse events and specifically weight gain. Weight gain induced by psychotropic medications can further compound psychological distress and negatively influence compliance. Here we report on four cases of social anxiety disorder treated with the atypical antipsychotic quetiapine. Self-administration of conjugated linoleic acid and green tea extract may have influenced objective anthropomorphic measurements; each patient had an unexpected decrease in total body fat mass, a decrease in body fat percentage and an increase in lean body mass. Since weight gain is a common and undesirable side-effect with psychiatric medications, our observation strongly suggests the need for controlled clinical trials using these agents. The prevalence of dietary supplement use has increased steadily over the last decade. Recent investigations show that 73 percent of US adults consume at least one type of dietary supplement during the course of a year . SimilarIn our outpatient clinic we have been inquiring on supplement use among those with mood disorders and anxiety for a number of years. In the interest of safety, we ask patients to disclose current dietary supplement use, and since many are taking, or will be initiating pharmacotherapy, we recommend discussion prior to patient's self-prescription with supplements.One significant area of dietary supplement use which can impact a psychiatric practice is that pertaining to weight loss. Patients with obesity-related depression and those attempting to address or prevent antidepressant or antipsychotic-induced weight gain are often attracted by the marketing of weight loss supplements. A recent audit of commercially available weight loss supplements in 73 different metropolitan retail outlets revealed 402 different products, with over 4000 separate ingredients purported to promote weight loss . For exaThe established potential of antidepressants and the emerging value of atypical antipsychotic medications in the mood and anxiety disorder are tempered by the well documented adverse effect of weight gain among users. In fact, the increases in body weight are not only significant, they can occur in a matter of weeks. For example, research shows that atypical antipsychotic medication can cause a ten pound weight gain in as little as eight weeks . Other sThe current observation involved four individuals whom we identified as suffering with the mood and anxiety disorders who described concerns related to weight gain and who disclosed that they would be co-initiating use of the previously mentioned green tea EGCG-CLA combination (abs+) upon prescription of their medications. All patients are routinely questioned on the current or planned use of dietary/herbal supplements in our clinic. While the EGCG-CLA combination is widely available in Canadian retail outlets, it was not sold, promoted, nor endorsed in our clinic. In a high-volume mental health practice, four patients disclosed intent to use the EGCG-CLA combination, and consented to objective weight-related measurements of skinfold thickness, before, during and at the time of discontinuing the self-prescribed EGCG-CLA combination. These four patients were not taking any other weight loss supplements and none made any dramatic changes to diet or physical activity levels.®) for periods ranging from 10–24 weeks. Patients reported co-self-administration of 270 mg EGCG, 3400 mg CLA daily in addition to the atypical antipsychotic medications and antidepressants they had taken for treatment resistant generalized anxiety disorders. Measurements of percent body fat were undertaken using the same method, first described initially by Durnin and Rahaman [Four adults with primary generalized anxiety disorder were treated with the atypical antipsychotic quetiapine  can significantly reduce food intake and body weight .Obviously this report documents only the changes in body composition among just four adults on atypical antipsychotics. Clearly, there are external issues which may have changed outcome, including the evaluation of patients who were motivated to self-administer a commercial supplement. This, coupled with the awareness of our interest in any subsequent anthropomorphic changes, could serve to alter other weight-related lifestyle factors. Still, we find the consistency of the results among all of those followed to be remarkable and one which raises the possibility of a real effect. We set out to follow these patients with the expectation that there would be only increases in body fat mass and body fat percentages, with little to no change in lean body mass.While no conclusions can be drawn, our observations should encourage a thorough scientific investigation concerning the utility of EGCG and CLA in the prevention of psychotropic medication-induced weight gain. Rapid weight gain among patients with mental disorders is an extremely distressing experience. In addition to the psychological fallout and potential non-compliance with otherwise effective medications, there is also the well known increase in risk of chronic diseases which accompany body fat accumulation. Every avenue of research should be exhausted in addressing side effects and medication-induced weight gain, including those related to the external products which mental health patients are self-prescribing.Within the past five years author Alan C. Logan has been a compensated independent consultant for Genuine Health Inc, the company which manufactures abs+. He holds no stock or shares and possesses no financial holdings with Genuine Health. The product used is not patented or under active patent application. There are no other competing interests to report among any of the authors.MK and LJ evaluated the subjects and performed the anthropomorphic measurements, MM and MV conceived of the value of these observations, participated in subject identification, coordination and performed data analysis. AL acted as technical expert with properties of EGCG and CLA and drafted the manuscript. All authors reviewed the data, assisted in the final manuscript construction and agree to its contents.


Listeria andSalmonella spp. can.In the past 10 years, evidence has suggested that group A streptococci (GAS) adhere to epithelial cells and invade them. In 1994, LaPenta and colleagues found that GAS could invade human epithelial cells, in some cases better than classical intracellular bacterial pathogens such asSeveral studies quickly confirmed this landmark finding and produced further evidence to support this mechanism. One study showed that high-frequency invasion needed expression of M protein  and/or fibronectin-binding proteins such as SfbI. Another study found that the M1 serotype of GAS was particularly adept at intracellular invasion of epithelial cells.It appeared, therefore, that GAS could invade human cells using several mechanisms, which suggested that intracellular invasion played an important role in the disease pathogenesis. The most direct evidence for intracellular invasion came from studies of patients with recurrent tonsillitis, when it was found that antibiotics failed to eradicate streptococci from the throat in about 30% of cases of pharyngotonsillitis. Osterlund and colleagues showed that tonsils excised from such patients contained intracellular GAS.The reason for invasion of host cells is not clear, although it was suggested that streptococci might find the intracellular environment a good place to avoid host defense mechanisms. Other theories suggested that internalization played a role in the carriage and persistence of streptococci. Internalization might also be involved in the invasion of deeper tissues, although some studies have found that low virulence was associated with internalization.PLoS Medicine, Pontus Thulin, Anna Norrby-Teglund, and colleagues try to further elucidate the mechanism behind GAS pathogenesis by examining the in vivo interactions between GAS and cells involved in innate immune responses. The team used human biopsies collected from 17 patients with soft-tissue infections. They found that host phagocytic cells, primarily macrophages, were being used as reservoirs for intracellular bacteria during the acute tissue infection.In this month'sThe data suggest that this could be a mechanism to avoid antibiotic eradication, which might explain why a high bacterial load was present even in tissues collected after prolonged intravenous antibiotic therapy. The authors also note that the localization of GAS varied and depended on the severity of tissue infection; intracellular localization was most frequent in noninflamed tissue, which also had a low bacterial load. Osterlund's study had already shown that intracellular GAS was present in pharyngeal epithelial and macrophage-like cells of patients with tonsillitis. However, the current study by Thulin and colleagues shows for the first time that this occurred in vivo during severe invasive GAS soft-tissue infections in humans, even while patients are on antibiotic therapy.Streptococcus iniae, which invades macrophage-like cells in a similar way. Studies are currently exploring whether this also occurs in GAS soft-tissue infections. But in any case, the clinical implication of this finding is that alternate therapies will be required if clinicians are going to improve the morbidity and mortality associated with severe GAS soft-tissue infections.The theoretical implication of this study is that if intracellular bacteria are most commonly found in newly involved tissue with low inflammation and low bacterial load, it might be the case that internalization could promote the spread of bacteria within the tissue. This so-called Trojan horse approach is also seen in the fish, and sometimes human, pathogen


Neuronal activity regulates gene expression to control learning and memory, homeostasis of neuronal function, and pathological disease states such as epilepsy. A great deal of experimental evidence supports the involvement of two particular transcription factors in shaping the genomic response to neuronal activity and mediating plasticity: CREB and zif268 . The gene targets of these two transcription factors are of considerable interest, since they may help develop hypotheses about how neural activity is coupled to changes in neural function.We have developed a computational approach for identifying binding sites for these transcription factors within the promoter regions of annotated genes in the mouse, rat, and human genomes. By combining a robust search algorithm to identify discrete binding sites, a comparison of targets across species, and an analysis of binding site locations within promoter regions, we have defined a group of candidate genes that are strong CREB- or zif268 targets and are thus regulated by neural activity. Our analysis revealed that CREB and zif268 share a disproportionate number of targets in common and that these common targets are dominated by transcription factors.These observations may enable a more detailed understanding of the regulatory networks that are induced by neural activity and contribute to the plasticity transcriptome. The target genes identified in this study will be a valuable resource for investigators who hope to define the functions of specific genes that underlie activity-dependent changes in neuronal properties. Transcription of new genes is initiated in the nervous system by both synaptic activity and action potential firing -3, and aVarious prior approaches have attempted to identify gene sets that underlie activity-dependent changes in neural function. cDNA microarrays have been frequently employed to characterize the plasticity transcriptome by identifying candidate genes that are upregulated after activity -15, typiIdentification of activity-regulated genes could be improved by genomic screens that are unbiased by cell type, target gene preselection, or average expression level. A growing body of knowledge regarding transcriptional regulation of gene expression and consensus sequences for transcription factor binding, combined with annotated genome sequences, makes it possible to carry out a directed search for binding sites of specific activity-regulated transcription factors at the scale of an entire genome. Methods for locating transcription factor binding sites often rely upon relatively simple comparisons of a single sequences or consensus binding sites with individual promoter regions -34. AlthHere we develop and employ a rigorous computational method for scanning gene promoters for binding sites of two known activity-dependent transcription factors: CREB and zif268. These transcription factors were selected based upon a large body of data that links their activity to adaptive responses to neuronal activity, especially activity occurring during learning . For exain silico analysis for full CREB and zif268 binding sites of promoter regions from all available annotated mouse genes. Likely binding sites were identified by applying position-specific scoring matrices (PSSMs) derived from previously characterized binding sites for CREB1 and zif268 confirmed by in vitro assays [We performed an o assays ,45. Indio assays .CREB sites have been demonstrated near both coding and non-coding regions of the genome and there is a growing body of data that supports an important regulatory role for CREB, and possibly other activity-dependent transcription factors, in non-coding regions . NonethAs a further test of the validity of these transcription factor binding sites, we performed this search in parallel on all human as well as rat genes with annotated transcription start sites. In general, because the rat genome is less well-annotated that either mouse or human, most analysis was carried out on mouse and human candidate genes. Dual hits from both the mouse and human genomes were considered more likely to have an activity-dependent component to their regulation. Our results identify 516 candidate genes with conserved CREB or zif268 binding sites in both mouse and human homologous genes that may be regulated by activity, six of which were predicted to have more than one type of transcription factor binding site. These results provide an important resource in understanding the regulatory networks that control activity-dependent programs of gene expression.We used a comparative genomics approach to identify genes likely to be regulated by neural activity. Position-specific scoring matrices were employed to create a search algorithm for DNA binding sites for CREB and zif268 in the promoters of annotated genes in order to define candidate genes from the plasticity transcriptome to guide and constrain future analysis. Gene lists were refined and evaluated by comparison of target frequencies across species and the presence of specific transcription factor binding sites in conserved homologous genes between human and mouse genomes. These gene lists allowed us to identify a significant subset of target genes that may be regulated in common by CREB and zif268. Finally, special attention was paid to the presence and relative frequency of genes with specific neural relevance amongst the candidate dataset, with an eye toward future experimental focus on the activity-dependant regulation of these genes.Previous analyses of transcription factor binding sites have suffered from low degeneracy based upon comparison of a target sequence to a single overrepresented binding site or high degeneracy and a high false positive rate based upon inclusion of related IEG subfamily members with similar but distinct sequence specificities. When related transcription factors  with overlapping binding site consensus sequences are pooled, binding site consensus is significantly relaxed and false positive rates are increased. Our method sought to reduce the rate of false positives by using a smaller number of experimentally verified, high-quality transcription factor binding sites using frequency matrices that have been experimentally developed for zif268 ,48 and CAn analogous search was attempted with AP-1, whose components, fos and jun, are also IEGs. The resulting predicted targets were not enriched for promoter regions and showed little conservation across species, facts we attribute to excessive degeneracy of the known AP-1 binding sequences used in training the computational methods. While we believe this negative result helps to reinforce the significance of the positive CREB and zif268 results, we have omitted further discussion of AP-1 in this report.In order to identify conserved gene candidates subject to activity-dependent regulation, the entire database of mouse, rat, and human promoter sequences was searched for CREB and zif268 binding sites. In total, 18,071 mouse genes, 5,943 rat genes, and 19,794 human genes were examined . In genTo develop a rigorous estimate of the quality of identified targets, we applied two methods for calculating their positive predictive value, defined as the probability a predicted hit is correct (see Methods for a complete description). This measure provides a conservative estimate of the percentage of identified transcription factor binding sites that we expect to be functional binding sites. For this purpose, we examined binding site location and cross-species conservation.Fig. Comparative genomics was used to provide a second method of estimating the positive predictive value. In the set of mouse-human homologues, 2.7% have a conserved CREB site and 1.2% have a conserved zif268 site. We proposed that the set of homologue pairs with conserved binding sites would provide a conservative set of true positive hits. The positive predictive value of the hits could then be estimated from the excess of conserved hits beyond what would be predicted from the hit rates in the individual genomes on the assumption of independence between genomes (see Methods). In contrast to the results using location specificity, the conservation approach yielded higher estimates of positive predictive value for CREB (0.83) than for zif268 (0.56). This difference is most likely to due changes in zif268 targets or promoter GC content between species. We can further validate these results by measuring conservation in binding site positions for the conserved hits. Comparing the positions of the conserved predicted sites between mouse and human yields correlation coefficients of 0.18 for CREB  and 0.21 for zif268 . There is considerable noise in location specificity, which might be explained by insertions or deletions in the promoters since the separation of human and mouse lineages or by errors in annotated transcription start sites in either species. The positions nonetheless show significantly greater correspondence than can be explained by chance.Prior analyses of activity-dependent gene expression have not addressed whether the transcription factors selected for analysis regulate separate or partially overlapping target genes. We reasoned that genes containing binding sites for both transcription factors might be more likely to have an essential role in mediating activity-dependent changes in gene expression. To examine this, we looked at the relative frequencies of genes with both CREB and zif268 binding sites in the mouse, human, and rat gene datasets compared to the frequencies of genes with either site in isolation. Target genes were generally non-overlapping, suggesting that each transcription factor may regulate a distinct subset of genes, a possibility that might allow a cell greater combinatorial control over stimulus-specific transcriptional programs.p < 0.05), but not significant for human (p = 0.18).Intriguingly, however, the amount of overlap between zif268 and CREB targets was greater than would be expected by chance, suggesting that at least a subset of target genes may be coregulated by the two transcription factors analyzed. Statistical analysis showed that the predicted binding sites shared more targets in common than would be expected by chance for both mouse and human genomes, with the results weakly significant for mouse , we chose to further this analysis using the species-specific gene target lists. We chose to present the mouse target list because the comparative data available has primarily been carried out in rodents. As with the conserved gene list, genes with CREB consensus sites showed significant overrepresentation for targets involved in RNA processing, but were underrepresented in electrophysiologically important transmembrane receptor targets  frequencies after 600 bp. This finding suggests that the functionality of these sites is greatest at proximal locations within the promoter, a conclusion that is further supported by the high conservation of location specificity between species. Our results strongly support the use of homology methods for improving specificity of binding site prediction ,70. Our Finally, this study has also led to some intriguing findings about the specific likely targets of CREB and zif268. Although these transcription factors are not exclusively expressed in the CNS – indeed, they are present in many other cell types at different developmental stages and regulate the transcription of gene targets that are not specifically neural – the essential role that they play in neural plasticity was an important consideration in motivating this study and the targets identified will be of strong interest to neurobiologists.Significantly, the study revealed a significant convergence of targets containing both CREB and zif268 binding sites. Among these putative CREB and zif268 co-regulated genes, the set conserved across mouse and human included several known transcription factors and transcription regulatory elements, most associated with AP-1 regulation. CREB and zif268 may represent the top level of a regulatory network implicated in neural function, with expression of the functional proteins primarily controlled by an intervening network of other regulatory factors. Indeed, it is clear from other experimental data as well as our own that additional waves of transcriptional regulation and distinct programs of gene expression will follow the activation of this initial cascade [Computational identification of putative targets of CREB and zif268 regulation has identified a set of likely direct targets of activity-dependent regulation that avoids biases inherent in current experimental methods for characterizing such sets. In addition to providing a candidate gene set for future analysis, the study has revealed a pronounced location specificity and bias for co-occurrence particularly in promoters of other transcription factors, which will be useful for improving detection algorithms and more completely characterizing the regulatory networks underlying activity-dependent gene expression.We compiled a database of gene promoter regions using sequences from mouse build mm6 , rat buiWhen searching for candidate genes, we defined a putative promoter to be the genetic sequence from -1,000 bp to +200 bp of each transcription start for human, mouse, and rat genes. A set of intergenic sequences was also compiled for human and mouse to construct a "random" control dataset of 1,200 bp sequences using the regions from -51,200 bp to -50,000 bp relative to each transcription start site, where the transcription factors CREB and zif268 are not likely to have regulatory function. Due to a decrease in sequence quality further away from transcription start, distal sequence regions were available for only 77% of total genes, leaving 13,475 mouse intergenic regions and 15,178 human intergenic regions far analysis  > P(Background)P(Model)) > log(P(Background))log(P(Model)) - log(P(Background)) > 0log(Position specific scoring matrices (PSSMs) or position weight matrices (PWMs), are a well-established method of motif finding ,76. We uS is the scoring matrix, A is the frequency matrix, n is the nucleotide, p is the position within each binding site. The pseudocount, b, is set at the relatively small value of 0.25 to allow limited tolerance of base-pairs which have never been observed in a given position for a binding site. When comparing this scoring matrix to a sequence of the same size, adding the scores for the nucleotide that is at that same position in the sequence gives you the log of probability that the sequence matches the model.where The probability that a sequence is not a binding site is based on background dinucleotide frequencies. For each individual species, we went through all promoters and calculated the probability of each dinucleotide transition. For instance, The goal of this study was to create a comprehensive list of possible transcription factor targets. The log of the sequence length is often subtracted to correct for the number of possible sites being searched. While subtracting by the full log of the sequence length would provide a more rigorous control, we deliberately chose to increase the sensitivity of the method at the expense of specificity. This increases the number of targets found while decreasing the average quality of the target genes, allowing us to better take advantage of homology to eliminate false positives. Instead of subtracting the log of the sequence length, we subtracted a smaller "correction value". The larger the correction value, the more stringent the search is. To analyze the effects of decreasing specificity on the quality of the target gene set, we use a measure called the positive predicted value. Intuitively, this measure is the probability that a predicted site is a true positive. The positive predictive value is defined as In terms of our data, the positive predictive value isThe expected number of sites is the number of sites expected to be conserved if there was no association between a binding site existing in mouse and its human homologue. A series of possible correction values are plotted against the positive predicted value ) - log(P(Background)) - logThe positive predictive values for the individual species is calculated by comparing the promoter region to the intergenic region . A higher specificity means a smaller number of higher quality binding sites are used. The predicted fraction of true positives or positive predictive value is defined as (true positives)/. This measure is estimated as (observed sites - expected sites)/(observed sites). The observed sites are the targets verified by comparative genomics while the expected sites are the number of binding sites one would find by chance if comparing independent human/mouse datasets.Click here for file


We review empirical evidence for the relations of recent and cumulative lead dose with cognitive function in adults.A systematic search of electronic databases resulted in 21 environmental and occupational studies from 1996 to 2006 that examined and compared associations of recent (in blood) and cumulative (in bone) lead doses with neurobehavioral outcomes.Data were abstracted after consideration of exclusion criteria and quality assessment, and then compiled into summary tables.At exposure levels encountered after environmental exposure, associations with bio-markers of cumulative dose (mainly lead in tibia) were stronger and more consistent than associations with blood lead levels. Similarly, in studies of former workers with past occupational lead exposure, associations were also stronger and more consistent with cumulative dose than with recent dose (in blood). In contrast, studies of currently exposed workers generally found associations that were more apparent with blood lead levels; we speculate that the acute effects of high, recent dose may mask the chronic effects of cumulative dose. There is moderate evidence for an association between psychiatric symptoms and lead dose but only at high levels of current occupational lead exposure or with cumulative dose in environmentally exposed adults. In the development of the adult lead management guidelines see , a numbein vivo K-shell X-ray fluorescence (KXRF) instruments].In reviewing studies of the health effects of lead, it is critical to understand the available lead biomarkers in terms of how they represent external exposure ; how they are influenced by metabolic factors ; and how the combination of these considerations affects inferences regarding the health effects of lead . We concin vivo KXRF were begun in some research laboratories in the 1980s, but it was not until the mid-1990s that reports began to emerge of KXRF-measured bone lead levels in relation to potential health indicators from epidemiologic studies with sufficient sample sizes  to have substantial statistical power. Thus, in this review we summarize all studies to date that measure cognitive function and both blood and bone lead levels (or acceptable surrogate for cumulative lead dose).Blood lead levels measured in epidemiologic studies with valid instruments and standardized calibration and quality control procedures have been reported in the literature for > 35 years. Bone lead levels measured by We begin our review with a discussion of three other reviews on the topic of lead dose and cognitive function . Balbus-Many methodologic issues of relevance to the epidemiologic investigation of lead and cognitive function have been addressed elsewhere in this minimonograph . When eva) contained no original research, b) were conducted on nonhuman subjects, c) were case reports, d) contained no standardized neurocognitive assessment outcomes, or e) lacked measures of both recent and cumulative lead dose.We conducted a systematic literature review of the association between blood and bone lead biomarkers and cognitive functioning in adults. Our aim was to select studies that compared markers of both recent and cumulative lead dose in their relations with cognitive function. Both occupationally and environmentally exposed adult populations were included. We searched the PubMed  and Psyca) exposure was assessed at an individual level; b) exposure was assessed with a biomarker; c) cognitive outcomes were objective, standardized tests; d) statistical adjustment for potential confounders including age, sex (in studies with both men and women), and education; e) data collection was similar in exposed and nonexposed participants; f ) time period of study was the same in exposed and nonexposed participants; and g) there was a detailed description of the approach to data analysis. We decided not to try to derive a pooled estimate across studies of the associations of lead dose biomarkers with cognitive function because of differences in methods for subject selection, blood and bone lead measurements, neurobehavioral outcomes, approach to regression modeling, and presentation of results across studies. Pooled estimates from metaanalysis also can be highly influenced by decisions regarding how and whether to pool certain results. We thus decided to present details for each study and discuss them in turn.We abstracted data from articles meeting the selection criteria. Study quality was assessed with the following criteria: a) environmentally exposed individuals in the general population, b) workers with current occupational exposure, and c) former lead workers without current occupational exposure to lead. We have summarized these studies in We identified three main types of studies that reported cross-sectional or longitudinal associations of blood and bone lead levels with cognitive function. These were of We identified six articles from three studies  that evaluated subjects with mainly environmental exposure to lead  and 2. On = 141) of NAS participants. This was subsequently followed up with a report on a much larger number of NAS participants  . Cross-sectional analyses in the original report found that increased blood lead levels across a relatively low range of levels [mean ± SD = 5.5 ± 3.5 μg/dL) were a stronger predictor, compared with tibia or patella lead levels, of poorer performance on tests of speed, verbal memory, vocabulary, and spatial copying skills. However, this was not confirmed in the larger, cross-sectional analysis, except possibly for scores on a vocabulary test (Four articles from the NAS reported associations of blood and bone lead levels in a cohort of older men. One of these articles  was a fis apart) . Cross-sary test . Conversary test  score ovary test .In a study of almost 1,000 persons 50–70 years of age randomly selected from the general population in the Baltimore Memory Study (BMS), a cross-sectional analysis showed that relatively low current blood lead levels were not associated with cognitive domain scores. However, moderate tibia lead levels (mean ~ 19 μg/g) were significantly associated with worse performance in all seven cognitive domains . Thus, iFifteen articles were identified of workers with current or past occupational exposure to lead. Eight of these studies used a surrogate measure of cumulative lead dose  rather than a direct measure of lead in bone. Among these studies, which compared blood and IBL lead dose, when the lead exposure was primarily current , most studies found an association between increasing blood lead values and worse cognitive function . Howevern = 803) found strong and consistent associations of blood lead levels with worse cognitive function after adjustment for covariates, but tibia lead levels were not as consistently associated (n = 57) in whom finger bone  lead levels were measured (One study of currently exposed lead workers in South Korea (sociated . The sammeasured . The secmeasured . This isa) tibia and blood lead levels are biologically related and blood lead is in equilibrium with bone lead stores; b) the error in measurement of tibia lead levels is larger than that for blood lead; c) controlling for cross-sectional associations could obscure longitudinal ones; and d) lead in blood reflects recent external exposure, and is in equilibrium with bone lead stores, possibly taking away explained variance from bone lead associations via this correlation in cross-sectional analyses.In the South Korean lead workers with current occupational exposure, a longitudinal analysis was performed to separate recent lead dose (measured as blood lead levels) from cumulative lead dose (measured as tibia lead levels), and acute effects from chronic effects in 575 subjects with complete data across the three study visits . The autResults of a cross-sectional analysis of former organolead workers showed that higher peak tibia lead levels  were related to poorer functioning on a number of cognitive tests, including those assessing manual dexterity, executive ability, verbal intelligence, and verbal memory . In a loSeveral lines of evidence suggest that increased blood lead levels are associated with psychiatric symptoms in adults, such as depression, anxiety, irritability, and anger. For example, a cross-sectional analysis of 107 occupationally exposed individuals showed increased rates of depression, confusion, anger, fatigue, and tension as measured by the Profile of Mood States POMS;  among thp < 0.05), as well as for the combined measure of all three BSI subscales .In occupationally exposed South Korean lead workers, tibia lead levels were significantly associated with more depressive symptoms measured by the Center for Epidemiologic Studies Depression scale CES-D;  after ada) may be a risk factor for cognitive decline, b) has risk factors in common with dementia, c) is an early reaction to declining cognition, and d) influences the threshold at which dementia emerges [for review see Psychiatric symptoms, specifically symptoms of depression, potentially share the same neural substrates with components of cognition, and thus may be important to late-life cognitive functioning. Compared with nondepressed elderly individuals, depressed elderly perform more poorly on tests involving attention, memory encoding, and retrieval. However, intelligence tests are more resistant to these effects of depression . DepressAPOE) ɛ 4 allele magnified the negative cross-sectional association of tibia lead levels with performance on the cognitive domains of executive ability, manual dexterity, and psychomotor skills . Other studies have found that APOE ɛ 4 modifies dementia outcome in individuals with previous traumatic head injury, suggesting that APOE ɛ 4 plays a role in recovery from brain insults . Last, studies of environmentally exposed adults who had notably higher exposures in the past suggest that bone lead level is more consistently associated with performance on cognitive tests than is blood lead level. The domains associated with lead dose do not differ in general by lead biomarker . The cognitive domains consistently associated with each biomarker in both environmental and occupational studies on adults include verbal and visual memory, visuospatial ability, motor and psychomotor speed, manual dexterity, attention, executive functioning, and peripheral motor strength. Comparisons of lead and psychiatric symptom associations in previously and currently exposed samples lend credence, although perhaps at higher thresholds than for cognitive outcomes, that neurobehavioral functioning is consistently associated with blood lead when exposure is currently high  and bone lead when exposure is primarily from past chronic exposure.Following is a summary of the findings from each of the three types of populations. First, cross-sectional studies of currently exposed lead workers showed that associations of blood lead levels and cognitive function were clearer than the associations for tibia, patella, or calcaneus lead levels, perhaps because the acute effects of recent dose in an occupational setting masked the chronic effects of cumulative lead dose. Second, previously exposed occupational populations demonstrated a stronger association between cumulative lead dose measured in tibia bone with cognitive deficits compared with blood lead levels. The two studies that deviated from these otherwise consistent findings may not have had sufficient power to detect any associations (These associations exist in multiple settings, including both occupational and non-occupational, in men and women, and in populations with diversity by socioeconomic status and race/ethnicity. This reduces the likelihood of associations by statistical chance or due to unmeasured confounding. However, this consistency cannot completely rule out the possibility of uncontrolled confounding or effect modification . In addiThe strength of associations between lead and cognitive function is strong and can be compared to the influence of age on cognitive function. The comparative magnitude of these effects has been reported in several studies. In currently exposed lead workers, cross-sectional associations showed that a 5-μg/dL increase in blood lead was equivalent to an increase of 1.05 years in age . The magLongitudinal analyses in the NAS observed that an interquartile range higher patella lead level was approximately equivalent to that of aging 5 years in relation to the baseline MMSE score  and an iLead has adverse effects on many other health outcomes in addition to cognitive function. This is not surprising given lead’s numerous biologic effects, including calcium agnonism and antagonism , bindingAssociations between lead biomarkers and cognitive outcomes have been demonstrated in both cross-sectional and longitudinal studies. In several of the longitudinal studies, change in cognitive function was explicitly modeled in relation to preceding lead dose or in relation to change in lead dose. In either case, the temporality condition is met. In addition, as bone lead is a measure that ascertains prior dose, even in cross-sectional analyses, analysis of bone lead with cognitive test scores evaluates lead dose that preceded current cognitive performance; thus, while cognitive assessment is cross-sectional, dose assessment is retrospective and cumulative. This again would minimize concerns about incorrect temporal relations.Nearly all reviewed studies found a dose–effect relation for blood lead, bone lead, or both. Existing studies do not allow determination of a threshold dose for either blood lead or bone lead or the shape of the dose–effect relationship at low dose levels. Associations have been observed in populations with mean blood lead levels as low as 4.5 μg/dL  and meanLead adversely affects the brain in a variety of ways. Lead is thought to increase oxidative stress, induce neural apoptosis, influence neurotransmitter storage and release, and damage mitochondria. The ability of lead to substitute for calcium allows it to affect calcium-mediated processes and pass through the blood–brain barrier. It may also interfere with zinc-dependent transcription factors, altering the regulation of genetic transcription . Animal Blood lead level is a measure of current biologically active lead burden and is therefore a better marker of the acute effects of recent lead dose. These are likely to be effects on neurotransmission and calcium enzyme-dependent processes such as synaptic plasticity. This could lead to circulating blood lead impairing, for example, information storage and retrieval mechanisms or processing speed, which have been suggested to impair performance on cognitive tests , 1996b. Lead may also influence cognitive function indirectly through its effects on blood pressure, hypertension, or homocysteine levels. Increased homocysteine levels, a well-known risk factor for cardiovascular disease, have also been associated with risk for poorer cognitive functioning  and riskAPOE genotype offers strong biologic plausibility to the inference that lead causes cognitive dysfunction (APOE ɛ 4 allele is a risk factor for lateonset Alzheimier disease (APOE ɛ 4 allele lowers the age of onset of the disease and accelerates age-related cognitive decline (APOE ɛ 4 is involved in the recovery response of injured nerve tissue (APOE ɛ 4 allele having reduced ability to promote growth and reduced antioxidant properties (APOE genotype with tibia lead level may be related to an impaired ability to counteract injury from lead exposure among APOE ɛ 4 carriers.We believe the effect modification by function . The APO disease , hippoca disease , and sen disease . It appe decline . Mechanie tissue , with thoperties . The intAnother recent study also offers biologic plausibility. In the former organolead workers, tibia lead level was associated with the prevalence and severity of white matter lesions on brain MRI, using the Cardiovascular Health Study white matter grading system . Tibia lThe removal of lead from gasoline, paint, and most other commercial products has succeeded in dramatically reducing environmental sources of lead exposure, and this has been reflected by the parallel declines in mean blood lead levels in Americans over the same time frame. However, lead has accumulated in the bones of older individuals, and especially those of lead workers exposed at the continued higher levels encountered in lead-using workplaces. Thus, past use of lead will continue to cause adverse health effects even when current exposures to lead are much lower than in the past. Lead in bone is not directly harmful to the central nervous system, and most of the structural and neurochemical damage is likely to have occurred decades ago. Nevertheless, lead in bone might serve as a source from which lead can be mobilized into blood, and potentially cross the blood–brain barrier. The chronic effects of lead may account for a proportion of cognitive aging; future research will be able to determine whether the chronic effects of cumulative lead dose alter the trajectory of normal cognitive aging. Research efforts should be directed to development of preventive interventions for both lead-associated cognitive decline with aging from past exposures, as well as the mobilization of current bone lead stores into the circulatory system leading to new health effects.Cognitive aging occurs in conjunction with the normal biological aging process. It remains to be determined whether lead affects cognitive aging in adults by permanently reducing brain circuitry capacity thereby lowering baseline cognitive functioning, or by inducing steeper declines in cognitive functioning, leading to abnormal cognitive aging. It may be that lead influences cognitive health through its relationship with depressive symptoms, hypertension, or homocysteine levels, all of which influence cognitive impairment and risk of dementia. Future investigations should explicitly account for these complex causal pathways, and also determine whether chronic effects of cumulative lead dose increases the risk for such clinically relevant syndromes as mild cognitive impairment .


Natural killer (NK) cells are involved in the antitumoral immunologic mechanism. These cells act through the release of cytotoxic molecules defined as NK cytotoxic factors (NKCF). Inhibitory factors of NK and NKCF mediated lysis have been described in in vitro assays. This study evaluates the induction of resistance to NKCF cytotoxicity by sera from 27 patients with colon, breast and lung cancer. Addition of these sera to the cytolytic assay where K562 cells and concentrated NKCF were used, induced resistance to NKCF mediated cytotoxicity in 21 cases (77%). The sera from the group with metastasis blocked NKCF lysis more markedly than the group with local tumours. However, no differences were observed when the groups with colon, breast and lung cancers were compared. This blocking effect was not found to be related to gamma interferon (IFN) levels. In a previous study, we described a tumour factor (NK-RIF) produced by human cell lines derived from metastatic adenocarcinomas. This factor blocked lysis of tumour target cells by NK cells. Consequently, it is proposed that the release of similar tumour factors with a capacity to induce resistance to NKCF may be involved in tumour growth and metastatic spreading in in vivo.


Molecular evolutionary studies of noncoding sequences rely on multiple alignments. Yet how multiple alignment accuracy varies across sequence types, tree topologies, divergences and tools, and further how this variation impacts specific inferences, remains unclear.Here we develop a molecular evolution simulation platform, CisEvolver, with models of background noncoding and transcription factor binding site evolution, and use simulated alignments to systematically examine multiple alignment accuracy and its impact on two key molecular evolutionary inferences: transcription factor binding site conservation and divergence estimation. We find that the accuracy of multiple alignments is determined almost exclusively by the pairwise divergence distance of the two most diverged species and that additional species have a negligible influence on alignment accuracy. Conserved transcription factor binding sites align better than surrounding noncoding DNA yet are often found to be misaligned at relatively short divergence distances, such that studies of binding site gain and loss could easily be confounded by alignment error. Divergence estimates from multiple alignments tend to be overestimated at short divergence distances but reach a tool specific divergence at which they cease to increase, leading to underestimation at long divergences. Our most striking finding was that overall alignment accuracy, binding site alignment accuracy and divergence estimation accuracy vary greatly across branches in a tree and are most accurate for terminal branches connecting sister taxa and least accurate for internal branches connecting sub-alignments.Our results suggest that variation in alignment accuracy can lead to errors in molecular evolutionary inferences that could be construed as biological variation. These findings have implications for which species to choose for analyses, what kind of errors would be expected for a given set of species and how multiple alignment tools and phylogenetic inference methods might be improved to minimize or control for alignment errors. Whole genome sequences of closely related species, such as those now available in mammals, flies, worms, yeast and bacteria, provide an opportunity for evolutionary analyses to greatly aid in this effort, but also present new challenges for sequence analysis [Annotation of analysis .The first step in studying the evolution of noncoding sequences is alignment. New tools have been developed for fast and accurate alignment of long stretches of genomic sequence (reviewed in -4) and bSophisticated molecular evolution models and tests have been developed over the last few decades to identify various forms of selection and sequence features, yet their application nearly always assumes a perfect alignment . It is cBecause of the inferential nature of evolutionary studies, no experiment in extant taxa could generate information about the true orthology of sequences, so simulations offer a tractable alternative. Molecular evolution simulations have been used to assess evolutionary analysis methods, including divergence estimation ,11 and pHere we present the results from a simulation-based study assessing the accuracy of multiple alignments and the effect of alignment accuracy on two fundamental evolutionary inferences: transcription factor binding site conservation and divergence distance estimation.cis-regulatory DNAs that contain functional binding sites for transcription factors and thereby control gene expression [The most frequent noncoding targets of comparative analyses are pression . Althougpression -34, theypression -42. PrecDivergence estimates inform nearly all evolutionary analyses. Accurate measurements of noncoding divergences are used for many purposes including differentiating functional from non-functional sequences based on constraint -51, showBelow we first examine multiple alignment accuracy across tools, sequence types, trees and divergences. We show that multiple alignment accuracy is primarily determined by the pairwise divergence of the two most diverged species. We next look at alignment accuracy of transcription factor binding sites. We show that although they align better than their surrounding noncoding DNA, they are misaligned at a high enough frequency such that precise studies of gain and loss events could easily be confounded by alignment errors. Finally we look at the impact multiple alignment accuracy has on divergence distance estimation. We show that divergences tend to be overestimated at short distances and cease to increase at a tool specific maximum divergence, corresponding to the point at which alignment accuracy reaches its minimum. We also show that overall alignment accuracy, binding site alignment accuracy and divergence estimation accuracy vary across branches in a tree such that terminal branches are aligned better than internal branches. Implications for method development and evolutionary analysis are discussed.For the purposes of this study we developed a molecular evolution simulator, CisEvolver, that incorporates several known characteristics of noncoding sequences. CisEvolver takes an ancestral DNA sequence and evolves it along a mutation guide tree, producing sequences for which we know the true alignment. The utility of such a simulation is that the sequences can be re-aligned using standard alignment tools and the accuracy of the tool alignment as well as the accuracy of any inference from the tool alignment can be measured by comparison with the true alignment. In cases where the error in an inference is due to both alignment error and error in the inference method itself, the contribution of alignment error to the total inference error can be directly measured by comparison of inference from the tool alignment and inference from the true alignment.We implemented CisEvolver with two types of sequences, background genomic sequence and transcription factor binding sites. Background genomic sequences are evolved according to the Hasegawa Kashina Yano 1985 (HKY85) substitution model , a PoissCisEvolver is freely available .Drosophila melanogaster [Using CisEvolver we simulated a large set of alignments on which downstream analyses were performed. Sequences were simulated over a range of total divergence distances on two, three and four species trees with fixed topologies and fixed branch length proportions as depicted in figure nogaster ,60 contanogaster . BindingAll alignments were performed using default parameter settings for Clustalw , Mavid , Mlagan Using simulated true alignments and tool alignments we characterized the variation in alignment accuracy across alignment tools, divergences and trees. Alignment accuracy was defined as the fraction of ungapped columns in a true alignment that were aligned identically in a tool alignment (see Methods & "sensitivity" in ). We exai. Alignment accuracy varies across tools and divergences figure .ii. The presence of transcription factor binding sites leads to higher alignment accuracy figure .iii. More species results in better accuracy when comparing trees of equal total divergence but different numbers of leaves figure .iv. The improvement of adding a fourth species is less than that of adding a third when comparing trees of equal total divergence but different numbers of leaves figure .v. Adding in-group species or out-group species to a pair of species has an insignificant effect on the alignment accuracy of the pair figures  &2F.In addition to these investigations into alignment accuracy across all species in alignments, we also examined the alignment accuracy for subsets of species within multiple alignments, attempting to relate the accuracy to the tree topology. We measured what we call leaf-to-leaf accuracy, node-to-leaf accuracy and node-to-node accuracy (see Methods). Leaf-to-leaf accuracy refers to the accuracy of the alignment of sister taxa  are consistent with the hypothesis that the pairwise distance between the two most diverged species in a tree effectively determines alignment accuracy. Across tools and divergences, adding ingroup or outgroup species to a pair of species of fixed divergence had an insignificant effect on alignment accuracy  figure  &2F. BruFinally, observation vi, that alignment accuracy varies across branches in a tree, is quite unexpected. The progressive alignment steps that these four tools use appear to be biased toward aligning leaf sequences better than internal nodes, where sub-alignments must be aligned figure . This biTo understand the relationship of the observed variation in alignment accuracy with phylogenetic analyses performed using automated alignments, we explored the following two evolutionary inferences.Using simulated true alignments and tool alignments of enhancers containing conserved transcription factor binding sites we examined the accuracy of binding site alignment and its relationship with overall alignment accuracy. We used two definitions of binding site alignment. Aligned sites were classified as either perfectly aligned, meaning every base in the binding site was aligned correctly across all species, or overlapping, meaning the binding sites across the species overlapped at at least one position (similar to definitions in ).We first looked to see if binding site alignment accuracy varies across tools and divergences. Indeed, across tools binding alignment accuracy is a decreasing function of divergence distance. Figure We next compared our two binding site alignment scores. We were somewhat surprised to see how different the two scores are, based on the intuition that conserved binding sites should make for good anchors and large indels in flanking sequences therefore ought to be the cause of most alignment errors. Instead it appears that binding sites are often still overlapping in an alignment even if they are not perfectly aligned. Figure We next looked to see how binding site alignment accuracy is related to overall alignment accuracy. Across tools, divergence distances and trees, binding site alignment accuracy is highly correlated with overall alignment accuracy, however, binding site alignment accuracy is consistently higher than overall alignment accuracy. Figure rho = 0.92 p < 10-10). We also looked at the length and average information content of binding sites to see if longer or more highly specified sites tend to align better. Across tools, divergence distances and trees, binding site alignment accuracy is correlated with binding site length (figure rho = 0.44 p < 0.3) and average information content (Spearman's rho = 0.40 p < 0.35) but neither correlation is significant, likely because of the small number of factors used in this study. Thus the greater the density and the longer and more specified the sites in an enhancer, the more likely the sites will be aligned correctly.Lastly, we looked at properties of enhancers and binding sites to see how they are related to binding site alignment accuracy. We expected that enhancers with a greater density of binding sites would align more easily. Indeed, across tools, divergence distances and trees, binding site alignment accuracy is strongly and significantly correlated with the density of binding sites in an enhancer  at short divergence distances but are always underestimated at long divergence distances. Figure Because divergence estimation accuracy appears to vary in different ways than alignment accuracy, we looked directly at their relationship. Figure We last looked to see if divergence estimation accuracy varies across branches in trees as alignment accuracy does. Across tools, divergence estimates were most accurate for leaf-to-leaf branches, less accurate for node-to-leaf branches and least accurate for node-to-node branches. Figure Molecular evolutionary studies of noncoding DNA have either relied on the intuition that closely related species can be aligned well or have ignored alignment error all together -4,9. To cis-regulatory code [Because gold standards for base-level noncoding and regulatory DNA alignment accuracy do not exist, we developed a simulation platform called CisEvolver that can evolve background noncoding DNA, transcription factor binding site DNA or a mixture of the two (enhancers). We implemented CisEvolver with features of background and regulatory sequence evolution that are well modeled and are present in most comparative systems. Certainly more complicated evolutionary phenomena have been observed, and in cases where modeling is successful, ought be the subject of future studies. For instance, substitution rates have been shown to vary across sequences and have been modeled in various ways, most commonly using a gamma distribution . In our ory code  and bindory code ,57 are dory code  for an iUsing alignments generated by CisEvolver we tested the accuracy of alignments generated by four commonly used genomic alignment tools. All tools were run using their default parameter values (see Methods). It is quite possible that the accuracy of the alignments generated by some of these tools is highly sensitive to parameter settings and scoring schemes. In this study we focused on consistent behavior across tools and also how variation influenced inferences and were therefore not concerned with the relative performance of each tool. In order for users to optimize the use of current tools and also in order for designers of alignment tools to understand which algorithmic innovations actually improve alignment accuracy (beyond parameter settings), a systematic analysis of parameters is needed. In this study, using just default parameters, we found that the primary determinant of multiple alignment accuracy is the pairwise divergence distance between the two most diverged species in the alignment figure . AlthougDrosophila pseudoobscura from Drosophila melanogaster, at 1.79 substitutions per site [The first evolutionary inference we examined was the measurement of the conservation of transcription factor binding sites in regulatory regions. Studies have used conservation of binding sites as either a means of classifying functional from spurious predictions -33 or foper site , accordiper site ,28,79 . The 10 kb sequences were evolved from the root node of the tree down the branches to leaves using a substitution and indel model. Substitutions occurred according to the HKY85 substitution model [D. melanogaster mono-nucleotide base frequencies and kappa set to 2.0 as has been observed in Drosophila [For the divergence estimation simulations, 100 simulations were run for each divergence distance. For each simulation, a 10 kb ancestral sequence was randomly generated from the on model , using tosophila . Indel epindel = 1 - eRk-R is the relative rate of indels to substitutions and k is the length of the branch. In Drosophila indels have been found to occur approximately 10% the rate of substitutions so we used R = 0.1 [D. melanogaster indel polymorphisms with a maximum of 58 bp [where  R = 0.1 ,85. Indeof 58 bp . Inserticis-regulatory regions that have been found to drive expression patterns in reporter assays recapitulating some or all of the expression pattern of an adjacent gene were collected from two recent papers on anterior/posterior patterning in D. melanogaster [D. melanogaster using BLAT [Thirty-six experimentally characterized nogaster ,60. The ing BLAT . A GFF fcis-regulatory regions used in the study have been reported to be bound or predicted to be bound by some combination of the following factors: Bicoid [The 36 : Bicoid , Caudal : Bicoid , Giant [: Bicoid , Hunchba: Bicoid , Knirps : Bicoid , Kruppel: Bicoid , Tailles: Bicoid  and Tors: Bicoid . Positio: Bicoid ,61 or we: Bicoid  using ME: Bicoid  (describ: Bicoid ). Matriccis-regulatory regions, PASTER [-3 for each of the eight PWMs. If sites were overlapping one was randomly chosen and the others were thrown out.For each of the 36 , PASTER  was usedcis-regulatory regions were evolved to each of the divergence distances. Sequences were evolved from the root down the branches of each tree using either a background or binding site mutation model. Non-binding site sequences in the enhancers were evolved according the HKY85 and indel models described above. Binding sites were evolved according to the HB98 substitution model [i in the binding site is given by:For the binding site conservation simulations, 25 replicates for each of the 36 on model . We haveon model ,57. The Q is the background substitution model (HKY85) and f is the PWM for the factor. Indel events were not permitted in binding sites and deletions from background sequences were not allowed to extend into binding sites.where Alignments were performed using default parameter values for each of the following tools: Clustalw , Mavid vAlignment accuracy was defined asCSU is the count of the ungapped columns in the simulated alignment and CTSU is the count of the ungapped columns in the simulated alignment that are aligned identically in the tool alignment. This measure is the same as "sensitivity" defined in [where fined in .CSU was the count of ungapped columns for which the alignment was joining either sequences or correctly aligned sub-alignments and CTSU was the count of such columns in the simulated alignment that were aligned identically in the tool alignment. For instance, in a four species alignment, the node-to-node alignment accuracy was only based on the columns for which Seq1 and Seq2 were aligned correctly to each other and Seq3 and Seq4 were aligned correctly to each other (figure Branch specific alignment accuracy was calculated similarly except that A script written in PERL that can calculate these measures is available for download .cis-regulatory regions and all replicates are reported. The Pearson correlation between the density of binding sites in cis-regulatory regions and each measure as well as the correlation between the length of binding sites for each factor and each measure were calculated using the R statistics package[Binding site alignment was evaluated based on two measures. Sites that had the same start and stop position in each sequence in an alignment were considered to be perfectly aligned. Sites that were overlapping by at least one base in each of the sequence in an alignment were considered to be overlapping. The fraction of sites that were perfectly aligned and the fraction of sites overlapping in alignments across all s package.Divergence estimates were calculated using the baseml program from the PAML package v3.14 . Baseml DAP designed the research, performed the research, analyzed the data and wrote the paper. AMM contributed to the development of the CisEvolver program. All authors contributed to the research design and the writing of the paper.Drosophila melanogaster release 4 coordinates [This file, in GFF2 format , containrdinates .Click here for fileThis text file contains horizontal counts matrices and vertical frequency matrices for each of the eight transcription factors used in this study.Click here for file


We would like to thank the correspondents for their thoughtful contributions to this important public health topic . As our Measuring TB transmission itself is difficult, as rates in staff are confounded by exposures outside the workplace, and mechanical air sampling techniques have had limited success. We have established a guinea pig air sampling facility to directly measure TB transmission in a hospital ward in Lima, Peru  and haveThe results of the current study cannot be generalized to regions too cold to tolerate enhanced natural ventilation and not every room may be as amenable to natural ventilation as the Peruvian rooms that we studied. However, the key conclusions are clear: high rates of natural ventilation were achieved even on days with little wind and even rooms without high ceilings and large windows were well ventilated, such that natural ventilation significantly exceeded mechanical ventilation.It is therefore clear that natural ventilation has an important role to play in the fight against institutional TB transmission in resource-limited settings. Mechanical ventilation is expensive to install, requires costly ongoing maintenance, may be dangerous if poorly maintained , and is clearly inappropriate for the great majority of resource-limited settings where the burden of TB is highest. TB infection control is an urgent priority, underscored by the emergence of extreme drug-resistant TB strains and the increasing congregation in potentially high-risk overcrowded settings of persons living with HIV through the roll-out of enhanced HIV care. When infectious TB patients share rooms with others, opening windows and doors to enhance natural ventilation is a simple, inexpensive, and effective strategy in the fight against nosocomial TB transmission.


Campylobacter. But after poultry farmers began using fluoroquinolones to treat respiratory disease in flocks, the drugs became less effective in people. In 2005, the FDA banned the use of fluoroquinolones on poultry farms because of these concerns. A new study now suggests that the ban may not be enough to fix the problem EHP 115:1035–1039; Price et al.][.Doctors use the fluoroquinolone class of antibiotics to treat food poisoning caused by Campylobacter, confirmed the bacterium’s identity using DNA analysis, then tested for antibiotic resistance using the minimal inhibitory concentration method.A team of Johns Hopkins researchers made weekly trips to Baltimore supermarkets for 20 weeks in 2004 and 15 weeks in 2006. Each week, they bought chicken from each of five different producers. Three producers had never used any antibiotics; two were major conventional producers that declared they had ceased all use of fluoroquinolones in 2002, three years before the FDA ban. The scientists tested one piece of chicken from each package for Campylobacter strains from the conventionally produced chicken were more likely to be resistant than the strains from antibiotic-free samples.The bacteria from conventional chicken were more likely to be fluoroquinolone-resistant than those from antibiotic-free products. The researchers compared each poultry producer to every other producer in a pair-wise fashion. In both 2004 and 2006, this statistical analysis showed that the Campylobacter was not decreasing in chicken from these producers, even after four years.In addition, between 2004 and 2006, the proportion of antibiotic-resistant bacteria on the conventionally produced chicken showed no significant change, indicating that the prevalence of fluoroquinolone-resistant The results suggest that once antibiotic-resistant bacteria have developed, they may show up on grocery store shelves long after drug use stops. The authors note that they could not verify claims of voluntary fluoroquinolone prohibition because poultry producers are not required to report their use of drugs in food animals to regulatory agencies.Campylobacter.Other studies have shown that resistant bacteria can linger in poultry farms’ water distribution and ventilation systems and in reused litter. The authors state that additional interventions, such as requiring thorough disinfection and regular litter changing in poultry houses, may be necessary to reduce the public health burden of fluoroquinolone-resistant