Mental and physical effects of energy drinks consumption in an Italian young people group: a pilot study.

INTRODUCTION: The primary consumers of energy drinks were athletes, to combat fatigue, but in reality, thanks to their fast expansion and economic growth, young adults and teenagers represent the new target market. Consumption of energy drinks by both recreational and competitive athletes has increased dramatically in recent years, though they are often unaware of what is being ingested, believing to improve their physical and psychological performance. The literature shows contradictions about the capacity of energy drinks to enhance psychophysical results. In relation to probable adverse effects induced by the irregular consumption of energy drinks, which in several cases are not so clear, we decided to investigate the possible relationship between the intake of energy drinks and the presence of mental and physical stress in young people and athletes. METHODS: Two experimental sessions, separated at least by 1 week, according to a randomized cross-over design, following this protocol were conducted: in the first session a mental and physical stress was conducted without the consumption of energy drinks, the second after energy drinks consumption. BAI (Beck Anxiety Inventory) and BDI (Beck Depression Inventory II) test have been used to test the mental stress, and a "cycle ergometer test" to test the physical stress. RESULTS: BAI and BDI tests results showed that before the consumption of energy drinks, subjects are considered in the range of "minimal level of anxiety", (10 and 60 percentiles) and do not report a level of depression. After the energy drinks consumption, a "mild level of anxiety" has been recorded, and the BDI showed a case with a pathological profile. The physical test recorded a small increase in the maximum heart rate was verified with the intake of an energetic beverage. CONCLUSIONS: The stimulating effect of Energy Drinks EDs on nervous system and cardiovascular system, must be checked and studied in deeper detail, because it may represent a risk for the health of young athletes.

Cold exposure increases exercise-induced oxidative stress.

AIM: We determined the combined effects of cold and exercise on oxidative stress during submaximal exercise. METHODS: Sixteen amateur male cyclists pedaled at a constant speed corresponding to 85% of maximal HR as determined in normal conditions. Eight athletes pedaled indoors at 23 °C while 8 athletes pedaled outdoors at a temperature of 4-6 °C. We then evaluated the levels of reactive oxygen metabolites and plasma levels of antioxidants after exercise. RESULTS: Performing a physical task in cold conditions increased the free radical production, as demonstrated by the augmented levels of reactive oxygen metabolites and the concomitant decrease of plasma levels of antioxidants in outdoors cyclists as compared to indoors cyclists. The overall ANOVA and the post-hoc comparisons revealed a significant exercise and temperature effect. The mean level of reactive oxygen metabolites in athletes who exercised indoors was significantly lower than that of the outdoor athletes. Moreover, the outdoors group presented plasma levels of antioxidants significantly lower than those of the indoors group. CONCLUSION: Since several sports are performed outdoors during the winter season, the increased risk of oxidative stress in cold conditions must be considered in these disciplines. Cyclists, football and rugby players, and runners are all affected by the elevation in oxygen radicals induced by cold and should take appropriate precautions, such as specific antioxidant integration.

Assessment of body fluid balance and voluntary drinking in ultimate players during a match.

AIM: Ultimate is a sport played by hundreds of thousands of people in more than 42 countries; however, it is still mainly known as a recreational more than a team sport, and further studies are needed to define its physical load. Particularly, since no studies relating Ultimate to hydration have been performed, we aimed to determine body fluid balance, voluntary water intake and the most reliable method for assessing the hydration status of players after a typical 80-minute Ultimate match. METHODS: bioimpedance, urine specific gravity and body mass changes to asses the hydration level of the players were measured. RESULTS: It was observed that not all of the methods are adequate to determine dehydration in Ultimate players, and that measurement of body mass changes represents a reliable and accurate technique. CONCLUSIONS: These findings demonstrate that ultimate as an intense sport that can induce significant fluid loss, which is not always replaced by individual drinking.

Oxidative stress and antioxidant changes during a 24-hours mountain bike endurance exercise in master athletes.

AIM: This work monitored changes in oxidative stress and antioxidant defence during an endurance exercise in over 40 years old athletes. METHODS: Subjects were monitored during the 24-hours mountain bike Idro Lake (North of Italy) competition which took place in June 2008. The race lasted for 24 h, starting at 10.00 a.m., ending at 10.00 a.m. of the following day and was based upon riding for as many kilometers as possible in the 24-hours time schedule in a 5.5 km circuit trail. The study included 6 men bikers, aged 44.8 +/- 2 years, who raced on an individual basis. Blood samples were collected and the oxidative stress was measured performing the d-ROMs test which determined the reactive oxygen metabolites (ROMs), whereas the antioxidant defence status was assessed determining the biological antioxidant potential (BAP test). RESULTS: The ROMs levels significantly increased after 8 h from the beginning of the competition (122 %), at the end of the race (162%), 24 h (158%) and 48 h (144%) post-race. The biological antioxidant potential significantly increased at the end of the race (128%) and remained elevated 48 h later (114%). After 72 h post-race, ROMs and BAP levels differed significantly amongst subjects, thus showing an individual response to oxidative stress. CONCLUSIONS: In conclusion, exposure to intense and prolonged exercise induced a marked increase in dROMs levels in master athletes, only partially counterbalanced by antioxidants in blood plasma. The long-term effects of oxidative agents on the human body requires further studies, but it is likely that a diet potentially rich in antioxidants would help preventing oxidative damage of body cells and tissues and enhancing recovering from the endurance performance.

Hypericum perforatum methanolic extract inhibits growth of human prostatic carcinoma cell line orthotopically implanted in nude mice.

The antiproliferative effect of serotonin-reuptake inhibitors (SSRI) and serotonin antagonists has been demonstrated in prostate tumors. Since Hypericum perforatum components act as serotonin-reuptake inhibitors and exert cytotoxic effects on several human cancer cell lines, in this work we analyzed the effect of a treatment with Hypericum perforatum extract (HPE) on the growth of human prostate cancer cells in vitro and in vivo. This study highlighted a significant reduction of tumor growth and number of metastasis suggesting that this natural compound may be useful in the treatment of prostate cancer.

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.

Mapping of mitochondrial metabolic competence by cytochrome oxidase and succinic dehydrogenase cytochemistry.

To map the mitochondrial capacity to provide adenosine triphosphate (ATP), the activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were respectively evidenced by diaminobenzidine (DAB) and copper ferrocyanide cytochemical techniques in the cerebellar cortex of adult rats. Sampling of the positive mitochondria was carried out by the disector procedure. The ratio (R) overall area of the precipitates due to COX activity within the single mitochondrion/area of the same organelle was automatically calculated to estimate enzyme activity vs mitochondrial size. The number of SDH-positive mitochondria/microm(3) of tissue (numeric density, Nv) was morphometrically calculated. Cytochemistry of key enzymes of the respiratory chain enables measurement of the actual capacity of individual mitochondria to provide ATP. This quantitative estimation allows morphofunctional mapping of the mitochondrial metabolic competence in discrete tissue and/or cellular compartments. (J Histochem Cytochem 49:1191-1192, 2001)

Quantitative immunohistochemistry of glucose transport protein (Glut3) expression in the rat hippocampus during aging.

Immunohistochemistry of Glut3 (45 kD), an integral membrane peptide mediating the transport of glucose in neurons, was carried out in the hippocampus of 3- and 28-month-old rats to assess the effect of age on energy metabolism. Free-floating sections of fixed-frozen hippocampi were processed for quantitative immunohistochemistry of Glut3. A rabbit affinity-purified antibody identified Glut3 immunoreactivity. Glut3 staining was intense in neuropil, axons, and dendrites, whereas nerve cell bodies were unstained. With aging, Glut3 reactivity was significantly decreased in the inner molecular layer of the hippocampal dentate gyrus (-46%) and the mossy fibers of the CA3 sector (-34%), whereas the stratum radiatum of CA1 did not show any difference due to age. These data document an age-dependent decrease in Glut3 expression in discrete areas of rat hippocampus. Glut3 constitutes the predominant glucose transporter in neurons and is found abundantly in regions with high synaptic density characterized by frequent bursts of function-adequate metabolic activity. Our findings therefore lend further support to the critical role of an impaired metabolism in age-related brain dysfunctions and disease.(J Histochem Cytochem 49:671-672, 2001)

Salt appetite in salt-replete rats: involvement of mesolimbic structures in deoxycorticosterone-induced salt craving behavior.

Chronic administration of the mineralocorticoid deoxycorticosterone acetate (DOCA) induces a steady and robust increase in salt appetite and plasma Na(+) over the course of treatment. Interestingly, salt appetite behavior persists in rats even with elevated plasma Na(+) levels. Since there is evidence that the pathways normally associated with salt and water homeostasis are relatively unaffected in the DOCA-treated rat, we hypothesized that other regulatory systems may be hyperactive giving rise to this dysfunctional condition. The mesolimbic dopaminergic system has long been associated with orienting and reward-seeking behaviors such as those observed in reproduction, drug abuse, and appetite. Furthermore, we have previously shown that chronic DOCA administration results in an increase in mRNA levels of the endogenous opiate enkephalin in male rats given 24-hour access to tap water and 2% NaCl (two-bottle choice). Thus, in the present study, we tested the hypothesis that the mesolimbic dopaminergic system is dysfunctionally sensitized to the presence of a salt stimulus in DOCA-treated animals. Four groups of rats were injected with DOCA (5 mg/rat/day, 11 days) and one with vehicle (all were given access to water but access to salt was regulated). Two DOCA groups were given 2 h of 2% NaCl access/day and on the last day, one group was not given access (2hX). One of the two remaining DOCA groups was given 24-hour access to salt (24h) and the other no access at all (24hX). Consistent with our hypothesis, in the shell of the nucleus accumbens (AcbSh) we found relatively higher enkephalin- and tachykinin-mRNA abundance in the 2h vs. 2hX and dynorphin-mRNA in the 24h vs. 24hX groups. In addition, there were decreases in dopamine transporter binding in the AcbSh and decreases in tyrosine hydroxylase immunoreactivity throughout the striatum in the 24h vs. 24hX group. Furthermore, rats denied access to salt (2hX and 24hX) had higher cholecystokinin-mRNA levels in the ventral tegmental area compared to the 2h and 24h groups, respectively. These results suggest that basal ganglia structures associated with reward and goal-seeking behavior may be activated to elicit salt craving behavior in the DOCA-induced salt-appetitive rat.

Impairment of rat thymocyte differentiation and functions by neonatal capsaicin treatment is associated with induction of apoptosis.

The direct and indirect interaction between the nervous system and its transmitters with the immune system was evaluated in the rat by using the neurotoxin capsaicin (Caps). In the present study we investigated the effect of Caps administration to neonatal rats on thymocyte subpopulation distribution and functions at different times after treatment. Caps treatment results in a marked reduction of thymus weight and cellularity. As shown by immunofluorescence and FACS analysis, profound depletion of double negative (DN), double positive (DP), and single positive (SP) CD4(+) cells was already evident at day 7 after treatment and persisted until day 28. Reduced numbers of SP CD8(+) cells were observed only at later time points. Analysis of TCR phenotype indicates that CD5(+) TCR gamma/delta(+) are particularly sensitive to neonatal Caps treatment. Caps-induced thymocyte depletion was associated with reduced proliferation in response to T cell mitogens. Moreover, in situ TUNEL reaction and agarose gel electrophoresis indicate that neonatal Caps treatment induces apoptosis of thymus cells. Morphological analysis reveals the presence of apoptotic cells in the subcapsular thymus cortical region. Overall our results suggest that Caps when administered at birth, profoundly affects T cell differentiation, likely through its ability to activate apoptotic cell death program.

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

The present study examined the effects of ageing on preprotachykinin-A (PPT-A) mRNA levels in discrete regions of the rat brain. Semiquantitative analysis of silver grains revealed a 16% statistically significant decrease in PPT-A mRNA in the shell of the nucleus accumbens (AcbSh), a 27.6% statistically significant lower level of PPT-A mRNA in the olfactory tubercle (Tu), a 19.2% and 31. 5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate-putamen (d-CPu) (v-CPu), respectively, a 30% statistically significant lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% statistically significant decrease in PPT-A mRNA in the habenula (Hb) and a 30% statistically significant decrease of PPT-A mRNA levels in the postero-dorsal part of the medial amygdala (MePD). No changes in PPT-A mRNA levels were found in the nucleus accumbens, core (AcbC), in the islands of Calleja (Icj), and in the medial preoptic area (mPOA). These results show that ageing of the central nervous system (CNS) is associated with widespread changes in tachykinin gene expression, suggesting that alteration in the tachykinergic system may have implications in the physio-pathology of the elderly.

Effects of adrenal steroids on basal ganglia neuropeptide mRNA and tyrosine hydroxylase radioimmunoreactive levels in the adrenalectomized rat.

To investigate the effects of type I (mineralocorticoid) and type II (glucocorticoid) receptor activation on striatal neuropeptide [preproenkephalin (PPE), preprotachykinin (PPT), and preprodynorphin (DYN)] mRNA and midbrain cholecystokinin (CCK) mRNA as well as striatal tyrosine hydroxylase radioimmunoreactivity (TH-RIC) levels, we administered either replacement levels of corticosterone (CORT; 0.5 mg/kg/day, s.c.) or pharmacological levels of deoxycorticosterone acetate (DOCA; a mineralocorticoid steroid with ability to bind to type I and type II receptors; 5 mg/kg, s.c.) to adrenalectomized adult male rats. After 1 week of recovery from adrenalectomy surgery, animals were injected daily with sesame oil or CORT for 1, 3, or 7 days or DOCA for 3 or 7 days and killed 16 h after the last injection. Adrenalectomy resulted in a decrease in all three striatal neuropeptide mRNA levels, compared with sham-operated rats. CORT replacement resulted in recovered PPE and PPT mRNA levels after 1 day and elevated PPE mRNA levels over those in sham-operated controls after 3 days. In contrast, DYN mRNA levels showed recovery after 7 days of CORT replacement. Results after DOCA treatment largely paralleled those after CORT replacement. There were no significant treatment effects on indirect markers of midbrain dopaminergic activity, i.e., CCK mRNA and TH-RIC. From these results we conclude that compared with striatal tachykinin and dynorphinergic neurons, enkephalinergic cells show greater sensitivity, whereas the dopaminergic system, including mesencephalic CCK, demonstrates an insensitivity to physiological CORT and to pharmacological DOCA treatment.

Does delirium contribute to poor hospital outcomes? A three-site epidemiologic study.

OBJECTIVE: To determine the independent contribution of admission delirium to hospital outcomes including mortality, institutionalization, and functional decline. DESIGN: Three prospective cohort studies. SETTING: Three university-affiliated teaching hospitals. PATIENTS: Consecutive samples of 727 patients, aged 65 years and older. MEASUREMENTS AND MAIN RESULTS: Delirium was present at admission in 88 (12%) of 727 patients. The main outcome measures at hospital discharge and 3-month follow-up were death, new nursing home placement, death or new nursing home placement, and functional decline. At hospital discharge, new nursing home placement occurred in 60 (9%) of 692 patients, and the adjusted odds ratio (OR) for delirium, controlling for baseline covariates of age, gender, dementia, APACHE II score, and functional measures, was 3.0, (95% confidence interval [CI] 1.4, 6.2). Death or new nursing home placement occurred in 95 (13%) of 727 patients (adjusted OR for delirium 2.1, 95% CI 1.1, 4.0). The findings were replicated across all sites. The associations between delirium and death alone (in 35 [5%] of 727 patients) and between delirium and length of stay were not statistically significant. At 3-month follow-up, new nursing home placement occurred in 77 (13%) of 600 patients (adjusted OR for delirium 3.0; 95% CI 1.5, 6.0). Death or new nursing home placement occurred in 165 (25%) of 663 patients (adjusted OR for delirium 2.6; 95% CI 1.4, 4.5). The findings were replicated across all sites. For death alone (in 98 [14%] of 680 patients), the adjusted OR for delirium was 1.6 (95% CI 0.8, 3.2). Delirium was a significant predictor of functional decline at both hospital discharge (adjusted OR 3.0; 95% CI 1.6, 5.8) and follow-up (adjusted OR 2.7; 95% CI 1.4, 5.2). CONCLUSIONS: Delirium is an important independent prognostic determinant of hospital outcomes including new nursing home placement, death or new nursing home placement, and functional decline-even after controlling for age, gender, dementia, illness severity, and functional status. Thus, delirium should be considered as a prognostic variable in case-mix adjustment systems and in studies examining hospital outcomes in older persons.

Preprotachykinin-A gene expression in the forebrain of Sardinian alcohol-preferring and -nonpreferring rats.

Increasing evidence suggests that TKergic mechanisms might play a role in ethanol intake control. Preprotachykinin-A (PPT-A) mRNA brain levels were measured in Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats. PPT-A mRNAs were about 50% lower in sP than in sNP rats in the bed nucleus of the stria terminalis (BNST), whereas levels in the olfactory tubercle (Tu) were about 30% higher in sP than in sNP rats. Our findings suggest that altered PPT-A gene expression might contribute to the different ethanol preference and intake of sP opposite to sNP rats.

In situ hybridization analysis of preprotachykinin-A and -B mRNA levels in short-term sodium depletion.

Tachykinins inhibit salt appetite when applied intracranially in a number of brain regions and may function as endogenous inhibitors of sodium intake. To test the hypothesis that induced increases in salt appetite might involve disinhibition via a reduction in endogenous tachykinin expression, we used a semi-quantitative in situ hybridization analysis to investigate changes in brain areas expressing preprotachykinin-A (PPT-A) and preprotachykinin-B (PPT-B) mRNAs of rats after 1 day of sodium depletion (1d Na dep). PPT-A mRNA levels were detected in neurons of the olfactory tubercle (Tu), the nucleus of the olfactory tubercle (LOT), the dorsal and ventral caudate-putamen (d-CPu and v-CPu), the bed nucleus of the stria terminalis (BNST), the medial preoptic area (mPOA), the habenula (Hb) and the postero-dorsal part of the amygdala (MePD). PPT-B mRNA levels were measured in fundus striati (FStr), d-CPu, v-CPu, BNST, mPOA, dorsomedial hypothalamic nucleus (DMD), arcuate nucleus (Arc), central amygdaloid nucleus (CeL), basolateral amygdaloid nucleus (BLV), LOT, Hb and basal nucleus of Meynert (B). 1d Na dep reduced by 33-61% the mean number of PPT-A grains/cell in Tu, LOT, d-CPu, BNST, mPOA, Hb and MePD compared to control animals. Levels of PPT-B mRNA were not reduced as much by 1d Na dep, although statistically significant reductions of 26, 34 and 17% were found in v-CPu, BNST and B, respectively. These findings, therefore, support the hypothesis that endogenous tachykinins exert an inhibitory influence over sodium appetite.

Regulation of preprotachykinin-A mRNA in genetic hypertensive and normotensive rats.

It is well-known that central administration of tachykinins (Tks) inhibit salt intake in rats. Recent studies have shown that conditions that arouse salt appetite, such as adrenalectomy and sodium depletion, induce a decrease in preprotachykinin-A (PPT-A) mRNA in discrete regions of the rat brain, suggesting that reduced levels of PPT-A mRNA in the brain may have a permissive role on the expression of salt appetite. It has also been shown that spontaneously hypertensive rats (SHR) show higher avidity for salty solutions than their normotensive control Wistar-Kyoto (WKY) rats. In this regard, the present study tested whether SHR and WKY rats differ in expression of the gene coding for PPT-A, the precursor for Tks peptides. Using semi-quantitative in situ hybridization histochemistry, we examined the level of PPT-A mRNA in discrete rat brain regions of SHR and WKY rats under no treatment, after 1 or 3 days of Na+ depletion. Levels of PPT-A mRNA were analysed in the olfactory tubercle (Tu), in the lateral olfactory tubercle (LOT), in the dorsal and ventral caudate putamen (d/v CPu), in the medial preoptic area (mPOA), in the bed nucleus of the stria terminalis (BNST), in the habenula (Hb) and in the postero-dorsal part of the amygdala (MePD). Semi-quantitative analysis of silver grains revealed a 27.5% lower expression of the PPT-A mRNA levels in SHR opposite to WKY rats under no treatment in v-CPu, mPOA, BNST and Hb. 1 day of Na+ depletion reduced PPT-A mRNA levels when opposite to Na+-repleted animals in Tu and mPOA in both SHR and WKY rats. On the other hand, when comparing SHR and WKY rats after 1 day of Na+ depletion, a 26% lower level of PPT-A mRNA was detected in Tu and d-CPu of SHR opposite to WKY rats whereas a 14% and an 18% lower level was detected in v-CPu and Hb, respectively. A lower expression of PPT-A mRNA in SHR compared to WKY rats was also found in BNST and MePD, although no statistical significance was detected in these two brain areas. In the last experiment, 3 days of Na+ depletion reduced PPT-A mRNA levels in mPOA while negligibly increased mRNA levels in d-CPu and v-CPu, in BNST, Hb and MePD, both in SHR and WKY rats. Conversely, when making comparisons between the two strains, a 35% lower level of PPT-A mRNA in SHR with respect to WKY rats was found after 3 days of Na+ depletion in d-CPu, v-CPu and mPOA. A lower gene expression, even though not statistically significant, was found in Tu, LOT, MePD. These findings show a consistent difference of PPT-A mRNA levels in discrete regions of the SHR brain opposite to WKY rats and confirm that 1 day of Na+ depletion reduces PPT-A mRNA in discrete brain regions. Since SHR are notoriously more salt-avid than WKY rats and Tks are potent inhibitors of sodium intake, the down-regulation of PPT-A mRNA may contribute to the higher natriophilia and, therefore, to the etiology of the hypertensive disease.

The natural history of delirium in older hospitalized patients: a syndrome of heterogeneity.

OBJECTIVES: To determine the presentation, course and duration of delirium in hospitalized older people. DESIGN: Observational cohort study. SETTING: Inpatient surgical and medical wards at a university hospital. PARTICIPANTS: 432 people over the age of 65. MEASUREMENTS: All participants were screened daily for confusion and, in those who were confused, delirium was ascertained using the Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Those who were found to be delirious were followed daily while in hospital for evidence of delirium. The Delirium Rating Scale (DRS) was used to describe the clinical characteristics of delirium. RESULTS: About 15% of subjects had delirium. Sixty-nine percent of delirious subjects had delirium on a single day. The DRS total was higher on the first day of delirium for those with delirium on multiple days than those with delirium on a single day (P = 0.03). Among those with delirium on multiple days, there were no patterns of change over time in specific DRS items. CONCLUSIONS: Delirium in hospitalized older people is common and has a varied presentation and time course. Clinicians and researchers need to consider this great heterogeneity when caring for patients and when studying delirium.

Effects of deoxycorticosterone acetate and diazepam on neuropeptidergic neurons in rat striatum.

The neurosteroid tetrahydrodeoxycorticosterone (THDOC) interacts with gamma-aminobutyric acid (GABA)/ benzodiazepine (BZ) receptors. To test the hypothesis that THDOC works partially through mechanisms associated with GABAA/BZ receptor function, deoxycorticosterone acetate (DOCA) and the benzodiazepine, diazepam (DZ), were administered short- (1 day) and long-term (11 days). Levels of mRNA for dynorphin, preprotachykinin and preproenkephalin in the striatum of adult male Sprague-Dawley rats were measured by in situ hybridization. Acute DOCA and DZ treatment produced parallel neuropeptide mRNA profiles, whereas chronic DOCA and DZ treatment yielded different patterns of neuropeptide gene expression. Chronic DZ treatment resulted in no significant increase in salt intake whereas chronic DOCA activated salt appetite. We suggest that acute DZ and DOCA interact with GABAA/BZ receptors; however, the results of chronic treatment suggest that DZ and DOCA operate through dissimilar mechanisms.