P = 0.002). However, the rate of in-hospital ventricular arrhythmias was similar across the range of admission potassium levels , Multivariate analysis showed that compared with “low-normal” potassium values, patients with “normal-very high” potassium levels experienced increased risk for 30-days  and 1-year all-cause mortality . In patients admitted with AMI, admission serum potassium levels of 4.45 to 5.2 mEq/L are not associated with in-hospital ventricular arrhythmias, but are associated with increased short and long-term mortality.Abnormal serum potassium levels are associated with an increased risk of ventricular arrhythmias and mortality in patients with acute myocardial infarction (AMI). The aim of the present study was to evaluate whether different levels of serum potassium, within the normal range, are associated with worse outcomes. The present study comprised 1277 patients with AMI and normal-range admission potassium levels (3.5–5.2 mEq/L), who were enrolled and prospectively followed up in the Acute Coronary Syndrome Israeli Survey between 2010 and 2013. Patients were divided into 4 quartiles based on admission potassium levels; “normal-low” (K ≥ 3.5 and K ≤ 3.9), “normal-moderate” (K > 3.9 and K ≤ 4.18), “normal-high” (K > 4.18 and K ≤ 4.45), and “normal-very high” (K > 4.45 and K ≤ 5.2). We analyzed the association between admission serum potassium levels and 7 days in-hospital complication rates, and 30-day and 1-year all-cause mortality rates. Patients with “normal-very high” potassium displayed increased frequency of baseline clinical risk factors and experienced a higher rate of acute kidney injury during hospitalization compared with the “normal-low” group (7.7% vs 2.4%;  In the early phases of acute myocardial infarction (AMI), the sympathetic nervous system is activated, as reflected by elevated levels of plasma catecholamines and modulation of β adrenergic receptor signaling. This activation leads to intracellular influx of potassium and decrease in serum potassium levels. Low potassium levels have been shown to increase the automaticity and excitability of myocardial cells, leading to the propensity for ventricular arrhythmias.Serum potassium levels play a major role in the outcome of cardiovascular (CV) events.,7–14 In a recent large retrospective study, the authors have found a U-shaped relationship between admission serum potassium levels and in-hospital morbidity and mortality among AMI patients. However, Madias et al did not find admission potassium levels to be a predictor of increased morbidity and mortality among patients with AMI.Several studies have previously illustrated the prognosticator role of profoundly low admission serum potassium, usually below 3.5 mEq/L, and the risk of in-hospital ventricular arrhythmias, among patients with AMI.Data regarding the association between admission serum potassium levels, within the normal range, and morbidity and mortality outcomes among AMI patients are still lacking. Therefore, in this study we evaluated the association between admission serum potassium levels, within the normal range, and early and late outcomes among AMI patients enrolled in the Acute Coronary Syndrome Israeli Survey (ACSIS).22.1 The study protocol was approved by the local institutional review board in accordance with the Declaration of Helsinki, and a waiver of consent has been granted to the investigators.Acute Coronary Syndrome Israeli Survey is a biannual national ACS survey that prospectively collects data from all patients admitted with ACS during a 2-month period from each of the coronary care units and cardiology wards operating in Israel. A detailed report of the survey design and methods has previously been published.2.2The complete ACSIS 2010 to 2013 survey database includes 1413 subjects. Only subjects with serum potassium levels within the normal values (3.5–5.2 mEq/L) at presentation were included in the current study. Subjects were excluded if their potassium level was lower than 3.5 mEq/L (n = 76) or higher than 5.2 mEq/L (n = 60). Thus, the final study sample comprised 1277 subjects.2.3Demographic, associated diseases, medical therapy, clinical presentation, admission blood pressure (BP) and heart rate values, electrocardiography, and baseline laboratory results were recorded on prespecified forms for all patients admitted with a diagnosis of ACS.In-hospital and 30-day outcomes data were ascertained by hospital chart review, telephone contact, and clinical follow-up data. Out-of-hospital 1-year mortality data were ascertained through the use of the Israeli National Population Registry. Data checks for completeness and consistency were based on computerized data queries issued at the coordinating center of the Israeli Association for Cardiovascular Trials and were completed from the medical reports attached to the form of each patient.2.4Hypertension was defined when a documented diagnosis was reported or when the patient was chronically prescribed antihypertensive medications. Diabetes mellitus (DM) was defined when fasting serum glucose was >126 mg/dL (7.0 mmol/L) on 2 separate readings, a history of DM was reported, or when insulin or oral hypoglycemic were taken. Smoking status was determined according to the questionnaire. Chronic renal failure was defined when a documented medical diagnosis was reported or when the calculated glomerular filtration rate was less than 60 mL/min. Congestive heart failure was defined when a documented medical diagnosis was reported or when the ejection fraction before admission was less than 40%. Dyslipidemia was defined by history, levels of cholesterol on admission and or use of lipids lowering medications.Patients were divided into 4 quartiles based on admission potassium values: “normal-low” (K ≥ 3.5 and K ≤ 3.9 mEq/L) (n = 336), “normal-moderate” (K > 3.9 and K ≤ 4.18 mEq/L) (n = 305), “normal-high” (K > 4.18 and K ≤ 4.45 mEq/L) (n = 321), and “normal-very high” (K > 4.45 and K ≤ 5.2 mEq/L) (n = 315).End points included 30-day and 1-year all-cause mortality.2.5For the univariate analysis, percentages were calculated for categorical variables and means with standard deviations for continuous variables. The linear-by-linear association chi-square test in case of categorical variables and the 1-way analysis of variance (ANOVA) test in case of continuous variables.For the study's 2 outcomes , we depicted Kaplan–Meier survival curves by potassium categories. The curves were statistically compared using the log-rank test for overall.Cox proportional-hazards models were used for the 30-day and 1-year mortality outcomes. The models were adjusted to prespecified baseline parameters; age, sex, glomerular filtration rate, hypertension, dyslipidemia, and DM.P-value calculations were 2-tailed and were considered statistically significant if their value was <0.05. The statistical analyses were performed with IBM SPSS version 22.0 .All 3The final study population comprised 1277 individuals, mean age was 64 ± 13 years and 991 (78%) were males. Mean follow-up was 482 ± 139 days. Compared with patients with “low-normal” potassium levels, patients with “normal-very high” potassium levels were older and had significantly higher rates of renal failure, DM, and previous myocardial infarctions (Table 3.1P = 0.002)  Fig. .3.2P value = 0.01 for the overall comparison among the 4 groups during 30-days of follow-up; Fig. Kaplan–Meier survival analysis showed that cumulative probability of all-cause mortality at 30 days was significantly higher among patients in the “normal-very high” potassium group (6%) as compared with the “normal-low” group (1%), the “normal-moderate” group (3%), and the “normal-high” group (3%)  compared with the “normal-low” group (4%), the “normal-moderate” group (5%), and the “normal-high” group (6%) .In the multivariate adjusted regression model, compared with patients with “low-normal” potassium levels, patients with “normal-very high” potassium levels had increased all-cause mortality at both 30 days and 1 year  and were conducted before the era of routine use of beta-blockers and early reperfusion therapies,,7–12 which have shown to reduce the incidence of postinfarction mortality and sudden cardiac death.,16 Interestingly, Choi et al have recently demonstrated that a U-shaped relation exists between mean serum potassium levels and 3-year mortality among patients with AMI. In particular, mortality was higher in patients with mean potassium levels above 4.5 mEq/L.Several studies have previously described the relationship of either profoundly low1 adrenergic receptor signaling abnormalities contribute to the development of arrhythmias. During AMI, β1 adrenergic receptor signaling is associated with cardiac hypertrophy, ventricular remodelling, and subsequently development of ventricular arrhythmias. Inversely, β2 adrenergic receptor activation has been found to attenuate cardiac remodeling in myocardial ischemic insult, thereby preserving cardiac function after AMI.,21 The occurrence rate of the majority of in-hospital complications, including ventricular arrhythmias, was found to be similar across all prespecified groups. This finding is of particular interest compared with previous reports, which emphasized the association of potassium imbalance upon admission and the increased risk for in-hospital ventricular arrhythmias.–11,13–15 This discrepancy might be explained by the fact that in previous studies, in-hospital morbidity and mortality were only significantly associated with extremely abnormal admission potassium levels, either profoundly low or high, whereas in our analysis, we have only included patients with normal-range potassium levels. Notably, Choi et al have also recently shown that there was no relation between mean serum potassium levels and the occurrence of ventricular arrhythmias.The β In addition, higher admission potassium levels (≥4.3 mEq/L) were also found to be associated with a larger scintigraphic infarct size in patients with ST-elevation myocardial infarction. These findings suggest that higher potassium levels, although in the normal range, may be associated with complications that are not necessarily related to immediate changes in the electrophysiological properties of the myocardium, and thus can be associated with late morbidity and mortality. For example, infarct size has been shown to be one of the main determinants of outcome in patients with AMI, even after 6 months. These findings provide a plausible explanation to our results, which demonstrated increased 30-day and 1-year mortality among patients with potassium values of 4.45 to 5.2 mEq/L, yet no increase of in-hospital complication rates, such as ventricular arrhythmias. It is possible that whereas normal range potassium levels are not associated with immediate detrimental conductive aberrancies, they are related to other structural and functional abnormalities which manifest later on, even after several months. Elevated admission potassium levels, even within the normal range, may be an indicator of severe sickness, thereby explaining the increased mortality. We indeed observed a higher rate of DM and renal failure in those with elevated admission potassium levels. Hyperkalemia occurs more frequently in patients with DM compared with nondiabetic patients.. The syndrome of hyporeninemic hypoaldosteronism (HH) is considered the most important causal factor for chronic hyperkalemia in DM patients, but other proposed mechanisms include hyperosmolality, insulin resistance or deficiency, and potassium-sparing antihypertensive medications. Notably, HH has been associated with increased risk of microvascular complications including diabetic nephropathy and neuropathy. Therefore, the increased mortality rates in patients with higher admission potassium levels observed in our study might be confounded by pre-existing DM and renal failure. However, the increase short and long-term mortality in these patients was significant even after adjustment for various comorbidities, including DM and renal function.The prognosticator role of normal range admission potassium levels among patients with AMI have been described in the past. Higher potassium levels (≥4.3 mEq/L) were shown to be an independent risk factor for target lesion revascularization among AMI patients.Our study has several limitations. It is an observational study and is restricted to Israeli adults, and as such it is unclear whether these findings could be generalized to other populations. We have only data on all-cause mortality and we do not know whether the increased mortality in patients with “normal-very high” admission potassium levels was related to CV causes. In addition, we do not have information regarding the long-term treatment of patients after getting discharged from the hospital, which could affect their survival and hospitalizations. Nevertheless, our analysis included a large number of patients from all intensive cardiac care units and cardiology wards in all public hospitals in Israel. The similar associations of the admission potassium level categories with 2 different end points strengthen the validity of our findings.In conclusion, our data suggest that admission potassium levels, even when in the normal range, might play a significant role in the risk stratification process of patients presenting with AMI.


This study uses production and perception experiments to explore tone 3 focus in Mandarin Chinese. Overall, contrastive focus in Mandarin is clearly marked with increased duration, intensity, and pitch range: in the experiments, listeners identified focused syllables correctly more than 90% of the time. However, a tone 3 syllable offers a smaller capacity for pitch range expansion under focus, and also yields less intensity increase; in addition, local dissimilation increases the duration, intensity, and pitch range of adjacent syllables within the same phrase as a focused tone 3 syllable. As a result, tone 3 focus was less well identified by listeners (77.1%). We suggest that the relatively poor identification of tone 3 focus is due to the smaller capacity for pitch range expansion, the confusion from within-phrase local dissimilatory effects, and the relatively weak intensity of tone 3. This study demonstrates that even within a language where purely prosodic marking of focus is clear, the location of prosodic focus can be difficult to identify in certain circumstances. Our results underline the conclusion, established in other work, that prosodic marking of focus is not universal, but is expressed through the prosodic system of each language. Focus represents the most important message in a sentence , a rising tone (tone 2), a low/dipping tone (tone 3), and a falling tone (tone 4). These tones are used to contrast homophonous morphemes, as illustrated in (1). Tones 1–4 are conventionally labeled as [55], [35], [214], and [51] depending on the pitch level, where [1] represents the lowest pitch level, and [5] the highest pitch level Chao, .(1) a. /ma/ with tone 1 ⇒ “mother”b. /ma/ with tone 2 ⇒ “hemp”c. /ma/ with tone 3 ⇒ “horse”d. /ma/ with tone 4 ⇒ “to scold” Xu, , p. 64Furthermore, Mandarin Chinese includes two basic kinds of pitch targets associated with tones: static and dynamic . In this case, although the entire sequence was in the domain of semantic focus, the tone 2 syllable seemed to carry main prominence (via focus).From a literature review, we observe several limitations in the stimuli of previous work. First, in many studies, a tone 3 syllable was excluded from the stimuli  Q: Has Tom got two or three watermelons?F you3 xi1 gua1.A: Bu4, [Ma3 Long2]“No, Marlon has the watermelons.”Li3 Min3) were designed to receive focus, but the sequence changed to Li2 Min3 due to tone sandhi. As a result, similar to the case of Greif  to determine the nature of tone 3 focus and its local dissimilatory effects; and (b) to examine whether listeners can successfully identify tone 3 focus or whether local dissimilatory effects hinder the recognition of tone 3 focus.Depending on the situation in which focus is used, it can be divided and labeled into several types, three of which are discourse-new focus, contrastive focus, and corrective focus. We designed stimuli that produced corrective focus within a paradigm of 10-digit phone-number strings and conducted production and perception experiments to achieve the goals of the current study.A Python script created randomized 100 10-digit strings, designed such that each digit occurs equally often in each position, and each pair of digits occurs equally often across each pair of positions. Please note that tone sandhi was avoided for target digits in the stimuli. The 100 10-digit strings were produced in two focus conditions: broad focus and corrective focus(3) a. Broad-focus condition李梅的电话号码是 787-412-4699。 “Li Mei's number is 787-412-4699.”b. Corrective-focus conditionQ: 李梅的电话号码是 887-412-4699 。是吗？ “Li Mei's number is 887-412-4699. Right?”787-412-4699。 “No, Li Mei's number is 787-412-4699.”A: 不是。李梅的电话号码是 Table We recruited two male and three female graduate students  at the University of Pennsylvania who had been in the US for less than a year at the time of recording. All the speakers were native Mandarin speakers with no speech or hearing impairments. Speakers signed a consent form and received 10 dollars as compensation.Recordings were conducted in a sound-attenuated booth at the University of Pennsylvania and were saved directly to a laptop using a Plantronics headset microphone at 44.1 kHz with 16 bits per sample. During the recordings, stimuli were presented to speakers through PowerPoint slides.Speakers were seated in front of a laptop monitor, wearing a headset microphone. In the beginning of the recording session, we presented speakers with three practice trials for each of the two focus conditions to familiarize them with the recording procedure. During the actual recording sessions, speakers first read 100 10-digit strings in isolation for broad focus, and (after a short break) they listened to pre-recorded questions through headphones and read the target strings as answers for corrective focus. The recording time was around 15 min for broad focus and around 25 min for corrective focus.In total, we collected 1000 10-digit strings (100 strings × 5 speakers × 2 focus conditions). Then, by dividing the strings by each tone, we collected 400 strings for tone 1, 100 strings for tone 2, 200 strings for tone 3, and 300 strings for tone 4. This particular breakdown of proportions is based on the fact that, as shown in Table Before analyzing the data, we illustrate some of the pitch contours that enable us to capture the overall picture of tone 3 focus, and then move on to the pitch contours that portray prosodic characteristics of each focused tone. In this study, the pitch contours were sampled at 10 equidistant points of each labeled digit using ProsodyPro Xu, .Figure Figure Based on the visual observations of Figures x indicates a raw f0 value in hertz (Hz), and 1 Hz is used as a reference value. In this study, we label the maximum and minimum pitch as “target pitch” for simplicity's sake. In pre- and post-focus positions, we measured duration (ms), mean intensity (dB), and mean pitch (st) from the positions immediately preceding and following focus to analyze the local dissimilatory effects of tone 3 focus. After carefully checking and manually correcting vocal pulse markings, we automatically obtained these measurements by implementing ProsodyPro -(NNN)-(NNNN), “N” refers to non-final position; “N” to final position; and “N” to a non-applicable position for pre-focus. Similarly, in the post-focus positions, since post-low bouncing also seems position-dependent, we divided the string position into two parts: initial vs. non-initial positions. In this string (NNN)-(NNN)-(NNNN), “N” refers to a non-applicable position for post-focus; “N” to non-initial position; and “N” to initial position.The basic analysis strategy was to make a direct comparison between the broad-focus and corrective-focus conditions, which are separated by focus position: focus, pre-focus, and post-focus. In the focus position, we examined the tone 3 digits by the aggregate measures of duration, mean intensity, and target pitch, and also included the other tones for reference data. In pre-focus positions, given that pre-low raising seems to occur only within the phrase, we divided the string position into two parts: final vs. non-final positions. In the 10-digit string  between groups. Here we present the results in the order of focus, pre-focus, and post-focus position. Please note that, for the pre-focus and post-focus positions, we will only provide the results of tone 3 focus since it produced a relatively poorer identification rate than that of the other focused tones.For our statistical analysis, we constructed a linear mixed model implementing the X2 = 64.10, df = 1, p < 0.001, R2 = 0.367; T2: X2 = 22.63, df = 1, p < 0.001, R2 = 0.319; T3: X2 = 47.92, df = 1, p < 0.001, R2 = 0.375; T4: X2 = 48.69, df = 1, p < 0.001, R2 = 0.319). Furthermore, the effect of focus on mean intensity was also significant for all tone types, indicating that corrective-focus conditions displayed a higher intensity than their broad-focus counterparts . Finally, focus produced a significant effect on target pitch for all tone types, resulting in corrective focus having greater pitch range expansion than broad focus .Figure lmerTest package . Given this finding, a post-hoc multiple comparison procedure was conducted to determine which tone type was significantly different; the output of the multiple comparison analysis is shown in Table X2 = 2.10, df = 3, p = 0.552, R2 = 0.378) and intensity , meaning that all tone types showed quite similar increases in marking prosodic focus, except pitch range expansion. Therefore, we speculate that the smaller pitch range expansion for tone 3 focus was due to its unique prosodic structure: tone 3 focus was expressed by lowering the pitch target, which distinguishes it from other focused tones we considered.Although the corrective-focus condition showed greater pitch range expansion than the broad-focus for all tone types, we must be sure to consider the smaller size of the pitch range expansion for tone 3 focus. As Table lmerTest . In order to further explore this result and to identify which position produced a significantly lower intensity, we conducted a post-hoc multiple comparison test. The results, as presented in Table Furthermore, it is worth noting that a tone 3 syllable produced the lowest intensity values in both broad-focus and corrective-focus conditions. The lowest intensity is deemed another unique characteristic of a tone 3 syllable in Mandarin. In order to test if this was so, we aggregated intensity values for each of the three focus positions: pre-focus, focus, and post-focusX2 = 16.62, df = 1, p < 0.001, R2 = 0.604) and mean intensity . Although non-final positions had greater mean pitch values than final positions, the effect of string position on mean pitch showed a positive trend but failed to achieve a customary level of statistical significance Moving on to consider the pre-focus position of tone 3 digits, Figure X2 = 11.25, df = 1, p < 0.001, R2 = 0.583; mean intensity: X2 = 6.77, df = 1, p < 0.01, R2 = 0.140; mean pitch: X2 = 20.22, df = 1, p < 0.001, R2 = 0.521). The results clearly suggest that tone 3 focus had different post-focus effects depending on the position in a digit string: the initial post-focus positions showed the post-focus compression effect, whereas the non-initial post-focus positions had the post-low bouncing effect.As for the post-focus position of tone 3 digits, Figure From the production experiment, we found that tone 3 focus was clearly marked by increased duration, intensity, and pitch range expansion. At the same time, we found that pre-low raising and post-low bouncing effects appeared only within the same phrase. Therefore, this perception experiment was aimed at examining whether listeners could successfully identify tone 3 focus or whether the local dissimilatory effects hindered the recognition of tone 3 focus.From the production data, we used 100 10-digit strings produced with tone 3 focus [2 tone 3 digits  × 10 string positions × 5 speakers], and also randomly selected 80 strings with other focused tones used for both distractors and reference data. The 80 strings were selected so that each tone had an equal number of focus tokens for each string position. A total of 180 strings were randomized and divided into two blocks of 90 strings each, and there was a short break between the two blocks.Twenty native Mandarin listeners were seated in front of a computer monitor and tested in a quiet room at Tongji University. The audio stimuli were presented to listeners through Sennheiser PC166 headset speakers using Paradigm software . We thus conducted a post-hoc multiple comparison analysis to determine which tone type received a significantly lower identification rate: Table Table As Table Let us now refer to tone 3 focus in Table The aims of this study were twofold: (a) to investigate the prosodic nature of tone 3 focus and its within-phrase local dissimilatory effects; and (b) to examine whether listeners successfully identify tone 3 focus, or whether the local dissimilatory effects present within the same phrase hinder the recognition of tone 3 focus. The method developed in this study allowed a systematic investigation of tone 3 focus and its within-phrase local dissimilatory effects. We have observed that production and perception results for tone 3 focus were compatible with each other.In production, tone 3 focus was realized with increased duration, intensity, and pitch range expansion similar to other focused tones. Some interesting phenomena with tone 3 focus included local dissimilatory effects—pre-low raising and post-low bouncing effects were present within the same phrase but absent across the phrase boundary. In perception, tone 3 focus received relatively low identification rates due to the interaction between local dissimilatory and phrase boundary effects when compared to other focused tones—incorrect answers most likely occurred in the immediate pre- or post-focus position within the same phrase.In this study, the key issue at hand was to ascertain why tone 3 focus achieved low identification rates. From the perception data, we observed that the local dissimilatory effects of tone 3 focus  clearly hindered the identification of tone 3 focus within the same phrase. In addition to these, there are at least two more reasons for the low identification rate: (a) smaller capacity for pitch range expansion; and (b) tone 3's low intensity by nature. We discuss these in turn below.First, lowering the pitch target results in a smaller capacity for pitch range expansion. Although other focused tones were expressed by pitch raising, tone 3 focus was expressed by pitch lowering. We assume that from a physiological point of view, there is more limitation on lowering the low pitch of tone 3 than raising the high pitch of other tones given that human pitch range is within 100 Hz  prosodic marking of focus is clear in Mandarin (e.g., Yuan, YL conducted a production experiment, analyzed the data and wrote the paper. TW conducted a perception experiment and collected the perception data. ML designed the experiment designs for both production and perception experiments and overviewed the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phalacrocorax auritus) for stable isotope ratios of carbon, nitrogen and sulfur. Cormorants exhibited discrimination factors that differed significantly among tissue types (for carbon and nitrogen), and differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in congeneric species. The Double-crested Cormorant has undergone rapid population expansion throughout much of its historic range over the past three decades, leading to both real and perceived conflicts with fisheries throughout North America, and this study provides an essential link for the use of stable isotope analysis in researching foraging ecology, diet, and resource use of this widespread and controversial species.The diet-tissue discrimination factor is the amount by which a consumer’s tissue varies isotopically from its diet, and is therefore a key element in models that use stable isotopes to estimate diet composition. In this study we measured discrimination factors in blood , liver, muscle and feathers of Double-crested Cormorants ( If we know ΔX and can measure δXt, we can solve for δXd, revealing information about the animal’s diet and foraging behavior. Discrimination factors vary among species and among tissue types within a species [The use of stable isotope analysis (SIA) has become widespread in studies of wildlife diet and foraging ecology, as the isotopic value observed in the tissue of an animal reflects that of its diet ,2. HowevδX = (Rsample—Rstandard)/Rstandard) x 1000 (equation 2), where X is, for example, 13C, 15N or 34S, and R is the corresponding ratio of heavy to light isotopes  in either the sample of interest (Rsample) or an international reference standard (Rstandard). Stable isotope ratios, including δ13C, δ15N and δ34S, have proven useful in identifying diet and resource use in wild animals. δ13C values are used in diet studies as an indication of the animal’s foraging environment as they are highly influenced by the type of photosynthesis by which carbon is fixed  as well as the source of carbon and the photosynthetic environment , and for this reason, information about primary production is largely preserved in δ13C values throughout the food web [Δ13C may be due to carbon isotopic fractionation during assimilation or respiration [Δ13C in a variety of aquatic and terrestrial food webs, and reported a range of Δ13C from -3 to 4‰, with averages of 0.2‰ [Stable isotope values are discussed here using delta notation: c, etc.; ). δ13C equation , where Xpiration . Researc of 0.2‰ , 0.4‰ [9δ15N values are often used in examining trophic dynamics within food webs as they exhibit significant Δ15N across trophic levels, and can therefore be used as an indicator of relative trophic position [Δ15N is generally positive, and is largely attributed to the excretion of isotopically lighter nitrogen in urine (or urate in the case of birds), leaving the isotopically heavier nitrogen within the consumer’s system to be assimilated through tissue growth. As with Δ13C, researchers have observed Δ15N in a variety of aquatic and terrestrial food webs, and reported a range of Δ15N from -1 to 10‰, with averages of 3.2‰ [position . Δ15N i of 3.2‰ , 3.4‰ [9δ34S values are highly influenced by whether sulfur fixation occurred in freshwater or marine environments [Δ34S among trophic positions, preserving information about the source of sulfur throughout the food web. Δ34S has been observed to range from -1 to 2‰, with an average of 0.2‰ [ronments , and, as of 0.2‰ . The maj of 0.2‰ ,13.Isotopic mixing models are increasingly used to identify diet composition . BecausΔ13C, Δ15N and Δ34S in the Double-crested Cormorant . The Double-crested Cormorant is a piscivorous colonial waterbird native to North America [In this study we identify  America . Over th America –19, lead America –25. Risi America –31. Thisad libitum diet of farm-raised channel catfish Ictalurus punctatus beginning the day after capture for a period of six or eight weeks (n = 3 and n = 3 respectively). Cormorants were sacrificed after the allotted feeding period, allowing for the collection of liver, blood  and feather (with active blood supply) samples. Samples of the catfish diet were also collected throughout the course of the study (n = 22). The duration of time on the catfish diet allowed the isotopic turnover of each tissue type discussed above [This research was conducted under the approval of the US Department of Agriculture, Wildlife Services, National Wildlife Research Center’s (NWRC) Institutional Animal Care and Use Committee (protocol QA-1723). Collection of specimens was permitted under USFWS Federal Fish and Wildlife Permit No. MB019065-6, and Mississippi and Alabama State scientific collecting permits. This study did not involve endangered or protected species. Six wild cormorants  were captured at night roosts in Lubbub Creek, Alabama, and Bluff Lake, Mississippi (MS) between 14 and 21 January 2010. These birds were transported to the captive waterbird facility at the NWRC Field Station in Starkville, MS. Cormorants were fed an ed above –34. Pected above ), the esδ34S and δ15N analysis without lipid extraction. Due to the influence of lipid content on δ13C in cormorant muscle and liver tissue [δ13C analysis. Lipids were extracted using a 2:1 chloroform:methanol solvent [All samples were frozen and transported to Cornell University for SIA preparation. Feather samples were rinsed with deionized water and dried. Muscle and liver samples were rinsed and drained of blood. Muscle, liver, blood and catfish samples were freeze dried. Muscle, liver and catfish samples were ground to a powder using a freeze mill. Half of each ground muscle, liver and catfish sample was set aside for r tissue , lipids  solvent .δ15N at the Cornell University Stable Isotope Laboratory (COIL) using a Thermo Finnigan Delta V Advantage isotope ratio mass spectrometer interfaced to a NC2500 elemental analyzer . Feather, blood, and lipid-extracted muscle, liver and catfish samples were analyzed for δ13C at COIL using EA-IRMS. An internal laboratory standard of American mink (Neovison vison) tissue was analyzed for every 10 study samples. A chemical methionine standard was used to measure instrumental accuracy across a gradient of amplitude intensities. Isotope corrections were performed using a 2-point normalization (linear regression) of all raw δ13C and δ15N data with two additional in-house standards: Cayuga Lake brown trout  and corn (Zea mays). Based on standard deviations of within-run replicate measurements of standards, analytical error was estimated to be ± 0.1‰ for δ13C, and ± 0.4‰ for δ15N. All non-lipid extracted samples were analyzed for δ34S at the University of Utah’s Stable Isotope Ratio Facility for Environmental Research using EA-IRMS. Internal laboratory standards were silver sulfide, zinc sulfide and eiderdown, and were analyzed for every 10 study samples. Based on standard deviations of within-run replicate measurements of standards, estimated analytical error was ± 0.5‰ for δ34S. Reference standards for 15N, 13C and 34S are atmospheric air, Vienna Pee Dee belemnite and Canyon Diablo troilite respectively.All non-lipid extracted samples were analyzed for p < 0.05. Discrimination factors were calculated for each cormorant tissue type by solving for ΔX using equation 1, where δXt is the mean isotopic value in each cormorant tissue and δXd is the mean isotopic value of the catfish. Standard deviation for discrimination factors was calculated by adding the standard deviation of isotopic values for each cormorant tissue type to that of the catfish such that it accounted for both sources of variation.All statistical analyses were performed in JMP version 9.0 (SAS Institute 2012). Analysis of variance (ANOVA) with post-hoc Tukey Kramer HSD test was used to identify differences in discrimination factors among tissue types. Statistical analyses were considered significant at δ13C value of -19.1‰ (±0.8‰), δ15N value of 7.6‰ (±0.5‰) and δ34S value of 2.0‰ (±2.3‰) and consisted of 44.2% C (±5.2%), 10.3% N (±1.0%), and 0.6% S (±0.1%). Δ15N values were all positive and generally large (ranging from 2.9 to 4.8‰). Δ13C values were smaller in magnitude (ranging from -1.1 to 0.2‰) and were predominantly negative. Similar to Δ13C, Δ34S values were small in magnitude  Δ13C and Δ15N; Δ13C and Δ34S values were observed, all were relatively close (within approximately 1‰) to zero. All discrimination factors observed in this study were within the range of values observed in freshwater aquatic food webs [Double-crested Cormorants exhibited discrimination factors that differed significantly among tissue types . Bearhop et al. [Unexpectedly, Double-crested Cormorant discrimination factors differed substantially (in the context of the isotopic variation among relevant prey species) from those observed in Great Cormorants  and stotelis ). While positive . It is lp et al.  used a cBecause of their substantial differences in magnitude and direction, use of discrimination factors from different species, even congeneric species like the Great Cormorant or European Shag, to model diet in the Double-crested Cormorant would introduce substantial error into model estimates, underlining the importance of using species- and tissue-specific discrimination factors in modeling efforts.


APOE) gene polymorphism, a known modulator of lipid metabolism, on plasma lipid levels in patients with hepatosplenic schistosomiasis.Schistosomiasis mansoni is a parasitic liver disease, which causes several metabolic disturbances. Here, we evaluate the influence of Apolipoprotein E , LDL-C, HDL-C and triglycerides. Schistosomiasis patients had reduced TC, LDL-C and triglycerides  compared to control individuals, whereas HDL-C was increased . Frequency of the common alleles, ε2, ε3 and ε4, was similar (P = 0.3568) between controls (n = 108) and patients (n = 84), implying that APOE genotype did not affect susceptibility to the advanced stage of schistosomiasis. Nevertheless, while patient TC and LDL-C levels were significantly reduced for each allele (except TC in ε2 patients), changes in HDL-C and triglycerides were noted only for the less common ε2 and ε4 alleles. The most striking finding, however, was that accepted regulation of plasma lipid levels by APOE genotype was disrupted by schistosomiasis. Thus, while ε2 controls had higher TC and LDL-C than ε3 carriers, these parameters were lower in ε2 versus ε3 patients. Similarly, the inverse relationship of TG levels in controls (ε2>ε3>ε4) was absent in patients (ε2 or ε4>ε3), and the increase in HDL-C of ε2 or ε4 patients compared to ε3 patients was not seen in the control groups.Blood samples were used for APOE gene polymorphism. Importantly, we also concluded that S. mansoni disrupts the expected regulation of plasma lipids by the different ApoE isoforms. This finding suggests ways to identify new metabolic pathways affected by schistosomiasis and also potential molecular targets to treat associated morbidities.We confirm that human schistosomiasis causes dyslipidemia and report for the first time that certain changes in plasma lipid and lipoprotein levels depend on  Schistosoma mansoni worms, is one of the most prevalent parasitic diseases. More than 200 million people are infected and worldwide at least 280,000 people die because of schistosomiasis every year, most in developing countries S. mansoni infections progress to hepatic fibrosis associated with portal blood hypertension Schistosomiasis, caused by S. mansoni infection reduces levels of plasma cholesterol and triglycerides in both rodents Previous studies have reported that human schistosomiasis alters plasma lipid composition APOE, gene; ApoE, protein), which distributes between triglyceride-rich lipoproteins  and high-density lipoproteins (HDL), helping to regulate their metabolism and the plasma levels of cholesterol and triglyceride. The APOE gene is polymorphic with three major alleles, ε2, ε3 and ε4, arising from point mutations at a single gene locus to produce three common protein isoforms, ApoE2, E3, and E4. The parent form, ApoE3 has cysteine and arginine residues at positions 112 and 158, respectively, while ApoE2 (Arg158Cys) and ApoE4 (Cys112Arg) have single amino acid substitutions One factor known to affect human plasma lipid concentrations is Apolipoprotein E .Eighty-four patients diagnosed with hepatosplenic schistosomiasis and attending the Gastroenterology Outpatient Department at the “Hospital das Clínicas - UFPE” were recruited during 2009 and 2010. The control group comprised 108 individuals with an epidemiological history incompatible with schistosomiasis and were drawn from the same age group (18–65 years) and socioeconomic background, as judged by a standardized questionnaire that enabled family budget, education level and lifestyle to be matched with those of the patients. Three stool samples from all individuals in both groups were also analyzed for parasitological infections. Subjects were excluded from the study if there was any evidence of parasitic infections, hepatitis B or C virus infections, cardiovascular or chronic kidney diseases, thyroid dysfunction or cancer. Individuals who had taken lipid-lowering drugs at anytime within the previous year were also excluded.Zona da Mata, an endemic area in the state of Pernambuco, northeast Brazil, and their grandparents and parents were also born in this same region. The study population comprised unrelated individuals. Hepatosplenic schistosomiasis was diagnosed by physical examination and upper abdominal ultrasound, conducted by a qualified and experienced professional according to the WHO protocol for ultrasound of schistosomiasis-related morbidity All participants lived in g (10 min at 4°C), stored at −20°C and used for lipid analyses within 24 h. Whole blood samples were stored at 2–8°C and APOE genotype determined within 7 days.Venous blood samples were drawn into evacuated tubes containing EDTA (0.562 M) after a 12 h fasting period. Plasma was separated within 2 h by centrifugation at 1500×Plasma total cholesterol (TC) and triglyceride (TG) concentrations were assayed by routine enzymatic methods. HDL cholesterol (HDL-C) was measured after precipitation of ApoB-containing lipoproteins from plasma with phosphotungstic acid in the presence of magnesium ions APOE polymorphisms (rs7412 and rs429358) were detected by polymerase chain reaction (PCR) HhaI (5 units/mL) for 3 h and the restriction fragments were separated by 4% agarose gel electrophoresis and stained with ethidium bromide (0.5 mg/L).Genomic DNA was extracted from leukocytes in whole blood, following a standard salting-out technique Genotyping was performed with blinding to subject identity. Sequence-proven controls were run with each PCR. A random 1/24 of samples were genotyped again on another day; no discrepancies were observed.2) goodness-of-fit test was used to assess deviation from Hardy-Weinberg equilibrium for each polymorphism and to compare categorical parameters among groups. All continuous variables were checked for normality and present a Gaussian distribution. Unpaired t-test was used to compare differences among continuous variables of SM patients and control individuals, while APOE allele groups were analyzed by one-way ANOVA followed by Fisher's Protected Least Significant Difference (PLSD). Lipid levels were adjusted for potentially confounding variables of age and gender. Pearson's Correlation test was used to estimate association between continuous parameters. Quantitative variables were expressed as mean ± standard error of media, while qualitative variables were expressed as absolute frequencies (percentage). P-values less than 0.05 were considered to be statistically significant. All statistical analyses were performed using StatView SAS Inc. .The chi-square , ε3 carriers (ε3/ε3 genotype) and ε4 carriers (ε4/ε4+ε4/ε3 genotypes). In each model, the homozygous ε3/ε3 genotypes formed the reference group. Six individuals  were excluded from the analyses because of the putative opposing effects of these two alleles.To evaluate APOE alleles among all participants were: ε2 – 11.46%, ε3 – 71.35%, and ε4 – 17.19%, similar to other studies in Brazilian populations 2 = 3.4164, φ = 3, p = 0.3318) and controls . Both control and patient groups showed similar mean age  and gender frequency  among the three different alleles. The allele frequencies were not statistically different between control and SM groups (P = 0.3568), indicating that APOE polymorphism was not able to affect the chance or course of schistosomiasis in this population.For this cross-sectional study, the two groups were matched by age and gender, as shown in P<0.0001) in the plasma levels of TC (25%), LDL-C (38%) and TG (32%). By contrast, the concentration of HDL-C was significantly increased in the patients   .APOE gene polymorphism on plasma lipid parameters, we repeated the analyses after subdividing each group on the basis of APOE alleles. Lower TC and LDL-C levels were found in the ε3 subgroup (ε3/ε3 genotype) of SM patients, as observed without allele differentiation (P from comparisons showed in To assess the influence of ntiation . Howeverntiation . This anP = 0.5360) in ε2 patient and control groups. This reflected a marked HDL-C increase (77%) and a positive correlation between TC and HDL-C  for patient ε2-carriers. By contrast, LDL-C was reduced in ε2 patients .Despite this significantly lower plasma TC in ε3 patients, the TC levels were similar  compared to their control counterparts ; and likDecreased plasma TG concentrations were seen in SM patients with the ε2 or ε4 variant alleles, but not for the ε3/ε3 genotype. However, the most striking difference in TG was noted in healthy ε4-carriers; their TG concentration was two-fold higher than the five other subgroups .APOE polymorphism, which influences the extent and nature of plasma lipid changes associated with schistosomiasis mansoni. In future studies, this finding will help in understanding how the parasite affects particular steps in host lipid metabolism and how host genetic background modifies disease progression and morbidity.Our report is the first to identify a host genetic factor, APOE genotype to influence infection susceptibility and damage in certain diseases caused by viruses, including human immunodeficiency virus APOE alleles are suggested to affect fibrosis progression in hepatitis C infection APOE gene polymorphism and schistosomiasis prevalence or severity.Several studies have shown the S. mansoni the decrease in plasma cholesterol is associated with a 50% reduction in atherosclerotic plaque progression, consistent with the low frequency of atherosclerosis noted in schistosomiasis patients The changes we report in plasma lipoprotein profiles, reductions in TC and LDL-C and an increase in HDL-C, is considered cardioprotective and hence can be regarded as a beneficial side-effect of schistosomiasis. We, and others, have previously reported low plasma total cholesterol in human studies S. mansoni eggs were responsible.Schistosomes need, but do not synthesize, cholesterol and one explanation for reduced host plasma cholesterol is that adult worms internalize LDL, via tegumental proteins analagous to mammalian LDL receptors S. mansoni cercariae resulted in reduced HDL-C Although TC and LDL-C were decreased in SM patients, we noted increased levels of HDL-C, consistent with an early report that alpha-lipoproteins were significantly higher in patients with Bilharzial hepatic fibrosis Effects of infection and inflammation on host lipoprotein metabolism are multi-faceted. Although the acute-phase response inhibits ApoAI synthesis and lowers HDL-C APOE polymorphism is well-documented to affect plasma TC and LDL-C; for example, meta-analyses by Bennet et al. APOE genotype on TC and LDL-C in our controls as mean values were significantly lower in ε2-carriers compared to the ε3/ε3 group , though unchanged for the ε4-allele. Interestingly, schistosomiasis abolished, and indeed reversed, this relationship; ε2 patients had higher mean TC and LDL-C than ε3/ε3 patients, although only the TC difference reached significance (P = 0.0458).In humans, APOE genotypes with HDL-C was reported by Bennet et al. S. mansoni infection and associated inflammatory responses and fibrogenesis. For example, we have reported LCAT deficiency in human The relation of S. mansoni infections markedly increase oxidative stresses We can speculate, therefore, that HDL-C increases in schistosomiasis are a multi-step process, promoted by low CETP activity and enhanced further by the ε2-allele: ApoE2 has a higher affinity than ApoE3 or ApoE4 for HDL S. mansoni infected mice had reduced hepatic expression of acetyl coenzyme A acyltransferase, an enzyme involved in fatty acid metabolism th week The plasma triglycerides change has inconsistent results in humans, as seen in mice earlier reports APOE genotypes with plasma triglycerides report non-linear relationships, the ε2- and ε4-carriers having higher levels than those with the ε3/ε3 genotype P<0.05 only for ε4-carriers; Meta-analyses to assess association of One limitation of this study is that it was conducted only at a single hospital, the Hospital das Clinicas, UFPE, which is the reference hospital for schistosomiasis in Pernambuco State, Brazil. Here, the Gastroenterology Outpatient Department receives the most severe cases of schistosomiasis, usually patients with a history of one or more episodes of gastrointestinal bleeding and hence most of the patients have the hepatosplenic form of the disease. Moreover, we had no information on plasma lipid levels before infection to compare with levels after the patients had developed the hepatosplenic form of schistosomiais. Therefore, the findings from the present study may not be extrapolated to all patients from other endemic areas who present with the hepatosplenic form of the disease.APOE gene polymorphism. Importantly, we also conclude that the normal regulation of plasma lipid levels by APOE genotype is disrupted by schistosomiasis mansoni. This finding merits further investigation; it may uncover new metabolic pathways and pathological processes associated with human schistosomiasis. In turn, these may identify molecular targets to aid treatment of schistosomiasis morbidity, and perhaps also inform other lipid-associated diseases, including atherosclerosis and diabetes.In summary, we confirm that human schistosomiasis causes dyslipidemia and, for the first time, report that certain changes in plasma lipid levels and lipoprotein profiles are dependent on patient


Rudbeckia hirta and R. fulgida. We then used several petal cut-and-paste experiments to test whether UV floral guides can also function to attract visitors. We manipulated floral guide size and evaluated visitation frequency. In all experiments, pollinator visitation rates were clearly associated with floral guide size. Diminished floral guides recruited relatively few insect visitors. Exaggerated floral guides recruited more visitors than smaller or average sized guides. Thus, UV floral guides play an important role in pollinator recruitment and in determining the relative attractiveness of conspecific flower heads. Consideration of floral guides is therefore important when evaluating the overall conspicuousness of flower heads relative to background coloration. This work raises the issue of whether floral guides serve as honest indicators of reward, since guide size varies in nature for conspecific flowers at the same developmental stage and since preferences for larger guides were found. To our knowledge, these are the first cut-and-paste experiments conducted to examine whether UV absorbent floral guides affect visitation rates and pollinator preference.Here, we report on the results of an experimental study that assessed the visitation frequency of wild bees to conspecific flowers with different sized floral guides. UV absorbent floral guides are ubiquitous in Angiosperms, yet surprisingly little is known about conspecific variation in these guides and very few studies have evaluated pollinator response to UV guide manipulation. This is true despite our rich understanding about learning and color preferences in bees. Historical dogma indicates that flower color serves as an important long-range visual signal allowing pollinators to detect the flowers, while floral guides function as close-range signals that direct pollinators to a reward. We initiated the work presented here by first assessing the population level variation in UV absorbent floral guides for conspecific flowers. We assessed two species,  A comprehensive, high quality literature exists regarding the sensory and cognitive abilities of bees. This research includes extensive data on essential tasks associated with foraging, like visual choice, learning and memory e.g. . Bees anThe color space perceived by bees has been determined using mathematical modeling and multidimensional scaling analysis that employed empirical color-choice test results . The colFor bees to be able to detect flowers in the distance, contrast between floral spectral reflectance and background reflectance is necessary e.g. . Green . Green cBombus L. terrestris). When foraging in a virtual flower meadow where ‘flowers’ had similar colors but different rewards, bees were more accurate when they took longer to make foraging choices  had higher pollination rates than white ones  had higher pollination rates than albinos (B. terrestris) have innate preference for high spectral purity  and showed that, when floral UV reflectance was obscured with sunscreen, fewer honey bees (A. mellifera scutella) approached and landed on flowers  flowers to successfully demonstrate their orientation function . First, we report novel findings regarding the distribution of floral guide sizes in natural populations and cultivars. We hypothesize that UV absorbent floral guides are important for attracting pollinators, not just for orientation, and that larger guides will be more attractive than smaller ones. To test these ideas we compare pollinator visitation rates after enlarging and diminishing natural UV-absorbent orientation cues. We focus on Halictidae (sweat bees), the relatively poorly studied, yet numerically abundant, cosmopolitan generalist pollinators frequently found in urban ecosystems  of the petal length for flowers from the first plant and 57% (standard error 0.8%) of the petal length for flowers from the second plant. These averages differed  despite the plants' shared environmental conditions. Floral guide size variation was greater between plants than for flowers on a single plant.n = 25, P<0.001). This simply means that bigger flowers had more UV absorptive petal surface area than littler flowers, which says nothing about the relative size of floral guides. Perhaps more compelling is the result that arises when we address the relative amount of the petal surface that is comprised of floral guide. No correlation existed between the flower head size and the proportion of UV absorptive petal surface area . In other words, larger flowers did not have relatively larger floral guides than smaller flowers.The average UV floral guide in the naturalized plants covered 51% (standard error 1.23%) of the total petal surface area. However, there was a 27% range in this estimate, with a min–max of 39–66%. A strong correlation existed between floral head size and floral guide size . Meaning once again, larger flowers did not have relatively larger floral guides than smaller flowers.The average UV floral-guide in the wild plants covered 44% (standard error 1.69) of the total petal surface area. This was 7% smaller than the urban population average guide size. However, there was a 39% range in this estimate, with a min–max of 26–64%. This range was 11% more than the urban population range. Like the urban population, no correlation existed between the floral head size and the proportion of UV absorptive petal surface area  = 50.1, P<0.001, two tail test). Post-hoc pairwise comparison tests indicated a difference between the number of visitors to the ‘sunscreen’ treatment when compared to the ‘water misted’ , and the ‘air sprayed’ treatment . The community of visitors was diverse and included the following 10 families: Scoliidae (n = 45), Apidae (28), Halictidae (18), Syrphidae (13), Megachilidae (13), Hesperiidae (2), Sphecidae (3), Culicidae (1), Muscidae (2), Vespidae (1) and an unidentified fly (1). Since Scoliidae are generally considered minor pollinators, removing this family, as well as the single visitors that were not likely major pollinators (e.g. Culicidae), and repeating the test did not affect the outcome. Visitors to the ‘sunscreen’ treatment included four families: Syrphidae (1), Megachilidae (1), Muscidae (2) and Vespidae (1).Sunscreen obscured the floral guide typically found on tion too . The fintion too . The Chi2 = 9.7, P = 0.008, two tail test). This result was not attributable to a positional effect of the treatments  = 0.7, P = 0.705). Post-hoc pairwise comparison tests indicated a difference between the number of visitors to the ‘enhanced’ treatment when compared to the ‘diminished’ treatment . Post-hoc results might appear counterintuitive, because the test is based on a squared deviation from the expectation. The squared deviation was smallest for the ‘enhanced’ treatment since it deviated least from the expected value, making the Δχ2 largest when comparing this treatment to the ‘diminished’ treatment because it had the greatest deviation from the expected value. The frequency of visitors to the ‘enhanced’ treatment increased over time but decreased for the control (after Day 1). Frequency of visitors for Days 1 to 6 to the ‘enhanced’ treatment were: 0.250, 0.140, 0.160, 0.375, 0.500, 0.625, and to the ‘control’: 0.633, 0.714, 0.600, 0.375, 0.375, 0.250).A total of 60 insects were observed visiting flowers in this experiment (23 visited the ‘enhanced’ (double ring) floral guide treatment, nine visited the ‘diminished’ (no guide) treatment, and 28 visited the ‘wild-type cut-and-paste control’; The community of visitors to this home garden experiment was less diverse than to the urban campus experiment. Here, Halictidae (sweat bees) comprised 75% (45/60) of the visitors and the number of their visits was similarly distributed across treatments each day. Substantially fewer individuals visited this garden from seven additional families: Papilionidae (3), Scoliidae (3), Apidae (2), Syrphidae (2), Megachilidae (2), Sphecidae (2), and Hesperiidae (1).2 = 8.2, d.f. = 3, P = 0.041). Post-hoc pairwise comparison tests indicated a difference in the number of visitors to the ‘diminished’ treatment and all other treatments . The community of visitors was diverse and included the following nine families: Apidae (33), Halictidae (33), Hesperiidae (9), Lycaenidae (6), Nymphalidae (6), Syrphidae (6), Pieridae (2), Hesperiidae (2), and Scoliidae (1).A total of 100 insects were observed visiting flowers in this experiment  floral guide treatment, 13 visited the ‘diminished’ (∼25%) floral guide, 26 visited the ‘wild-type cut-and-paste control’, and 30 visited the ‘natural’ flower; 2 = 13.6, P = 0.0011). Post-hoc pairwise comparison tests indicated a difference in the number of visitors to the ‘enhanced’ treatment compared to the ‘wild-type cut-and-paste control’ and ‘natural’ flower . The total visitation times were 472 seconds to the ‘enhanced’ treatment, 50 seconds to the ‘wild-type cut-and-paste control’ and 62 seconds to the ‘natural’ flower. Mean visitation times were 23.5 (s.d. = 27.0), 10.0 (s.d. = 9.59) and 10.3 (s.d. = 9.11) seconds, respectively. ANOVA analysis indicated that mean visitation times were not significantly different (Sums of Squares (SS) between groups = 8.8088, SS within groups = 70.54, Total = 78.6, d.f. = 2, 27 and 29, respectively; Mean Square (MS) between groups = 4.04, within group 2.61, F = 1.548 and P = 0.231). The community of visitors decreased in diversity and included two families: Halictidae (30), and Apidae (1).In June, a total of 31 insects were observed visiting flowers in this experiment (20 visited the ‘enhanced’ (90%) floral guide, five visited the ‘wild-type cut-and-paste control’, and six visited the ‘natural’ flower; 2 = 8.3, P = 0.016). Post-hoc pairwise comparisons again indicated a difference in the number of visitors to the ‘enhanced’ treatment compared to the ‘wild-type cut-and-paste control’ , but not to the ‘natural’ flower . The total visitation times were 192 seconds to the ‘enhanced’ treatment, 75 seconds to the ‘wild-type cut-and-paste control’ and 71 seconds to the ‘natural’ flower. Mean visitation times were 10.6 (s.d. = 13.6), 8.22 (s.d. = 7.34), and 14.6 (s.d. = 20.0) seconds, respectively. ANOVA analysis indicated that mean visitation times were not significantly different . The community of visitors included three families: Halictidae (17), Apidae (12) and Syrphidae (3).In September, a total of 32 insects were observed visiting flowers in this experiment . Post-hoc pairwise comparison tests indicated a difference in the number of visitors to the ‘enhanced’ treatment compared to the ‘natural’ flower  but not to the ‘wild-type cut-and-paste control’ . The community of visitors increased slightly in diversity compared to the two prior experiments due to an influx of butterflies and included five families: Halictidae (15), Nymphalidae (2), Pieridae (2) Papilionidae (1), and Apidae (1). The one-day food color mark recapture study yielded no repeat visitors on Day 2 but captive insects retained their color.In October, a total of 21 visitors were observed visiting flowers in this experiment . Chittka et al. label this reflectance pattern ‘green’ for bees . Chittka et al. label this pattern ‘UV-green’ for bees . Bees are capable of learning fine color differences through trial and error . In Image J, a phenotypic trait, like petal surface area can be traced and digitized. The circumscribed area can be calculated and the resultant pixel measurement for the trait can be converted to a meaningful scale (e.g. mm2) based upon a reference scale used in the picture . Multiple traits were measured including petal length, petal surface area, floral-head area, UV floral guide length, and proportion of petal surface area comprised of UV floral guide. All statistical tests were performed in SPSS.UV photography was conducted using a Baader U-filter. This filter transmits UV wavelengths from 325 to 369 nm, with maximal penetrance in the UV-A range at 360 nm . The filR. hirta seeds were obtained from the Ornamental Plant Germplasm Center of The Ohio State University. In March 2011, 236 seeds were planted under full-spectrum 12L: 12D lighting in the Kaplan Orchid Conservatory of Old Dominion University . After seven weeks, plants were transferred the ODU greenhouse until flowering. In May 2011, flowers were used in a pilot study assessing UV-absorptive floral-guide length (mm) variation. Since plants were grown in the same conditions, variation may represent the genetic component of phenotypic diversity. Floral guides were measured for 18 flowers and two plants and these measurements were compared with an independent samples t-test. The t-test assumption regarding equal variance was met .In November 2010, native 2) relative to the entire petal surface area, for each flower. We also evaluated the distribution of these guide sizes for this naturalized R. hirta population. Measurements were made for 30 flowers using Image J on photographs of all flowers. Flowers measured were located in a large, conspecific population on the ODU urban campus.In July 2010, we assessed the average sized UV absorptive floral guide by measuring the proportion of petal surface area that was comprised of floral guide .2), because this allows us to address questions like ‘Is UV guide size correlated with overall flower size?’. Since a significant result could merely represent a positive correlation between the total floral head size and amount of petal surface area that was floral guide, we next considered whether there was a correlation between the total floral head size and the proportion of the total petal surface area that was comprised of floral guide. This allows us to address whether larger floral heads have relatively larger guides  than smaller floral heads.Next, we used Pearson's correlation coefficient to determine whether there was a relationship between the floral head width (in mm), and the total size of the floral guide  were evaluated in the same manner as the naturalized urban flowers. Based upon the Shapiro-Wilks' test, the shape of the distribution of UV floral guide sizes for this population, like the urban one, was also normally distributed .R. hirta and R. fulgida) to determine whether insect visitation rates differed when floral guides were removed, enhanced and diminished. No flowers were reused in any trials.In the following series of experiments, floral guide size was manipulated in one of two species  to mask the floral guide entirely. Treatment two was ‘water misted’ as a control for moisture (the true sunscreen control of sunscreen minus the active ingredient was unavailable). Treatment three was ‘air-sprayed’ as control for potential loss of pollen during water misting.In July and August 2010, we conducted a sunscreen study with R. hirta (12 m×1.5 m), a smaller tract of Echinacea purpurrea (5′×2′) behind the R. hirta, and a few Hosta cultivars on the garden edges.This study site was intentionally selected to assay the community of insects naturally attracted to cultivars in a large, landscaped urban garden. The garden was situated in front of a five-story building and comprised of a large tract of All trials were completed on hot summer full-sun days a few days apart from one another. At ∼10:00 am, one hour trials were initiated and the total number of insect visitations was recorded. To be counted, the insect had to alight on a flower head then demonstrate some visual evidence of foraging or pollen collection. First landings were the sole landings counted for the few insects that were observed landing twice so as to avoid repeated measures. Insects were identified for this and subsequent experiments based upon our own expertise and guidebooks e.g. . VisitatR. fulgida plants surrounded by a variety of additional flowering perennial species. Treatment R. fulgida (Goldsturm) cultivars from a chain store were used in these experiments to evaluate insect response to experimentally diminished and enhanced floral guides.In August 2010, the first of a series of ‘cut-and-paste’ experiments was conducted in a diverse urban home garden (∼6 m×6 m) in Norfolk, VA. This garden was more complex than the above one, comprised of scores of cultivated To create the ‘cut-and-paste’ treatments ∼10 individual floral heads were collected for each trial. All petals were removed from heads, then cut sagittally at the point where the UV floral guide ended . UV photographs were taken to determine whether subsequent trimming was necessary to ensure that pieces were entirely floral guide or completely devoid of floral guide. The appropriate petal pieces were glued onto fresh floral heads contingent upon treatment.Two ‘cut-and-paste’ treatments and one control were freshly constructed for each trial. Treatment one had a ‘diminished’, or nearly no, floral guide and was created by gluing the petal pieces devoid of floral guide atop the proximal portion of an unmanipulated flower head. Treatment two had an ‘enhanced’ or double floral guide, where an additional concentric ring of floral guide was added distally to the natural guide by pasting small floral guide petal pieces on another unmanipulated flower head. For the ‘wild-type cut-and-paste control’ flower, two sets of petal pieces, one with floral guide, and one devoid of it, were glued atop the petals of a final unmanipulated floral head, recreating the common wild-type pattern.Each floral head  was placed into a clear glass bottle on the ground ∼3 meters away from the conspecific flowerbed. Six trials were conducted from about noon until 2:00 pm for six days and each ended after 10 insect visitations. Treatment locations were rotated in the bottle line-up in subsequent trials  such that each trial was comprised of a novel treatment series. Trials were conducted from August 21–27 in Norfolk, VA . The time, treatment visited, weather, and visitor type were recorded. Visitation data were analyzed with a Chi-square test and a post-hoc pairwise comparison test, as above.R. fulgida were again altered to create two treatments and one ‘wild-type cut-and-paste’ control. Treatment one was ‘enhanced’, here to be slightly enlarged from the natural floral guide to ∼60% of the petal surface area. Treatment two was ‘diminished’, here to be ∼25% of the petal surface. Treatment three was the same ‘wild-type cut-and-paste control’ used previously. Additionally, a ‘natural’ unmanipulated flower head was added as a true control for our ‘cut-and-paste’ procedure. Treatment bottles were positioned near the Rudbeckia plot of the ‘meadow flower bed’ at Weyanoke Bird and Wildflower Sanctuary in Norfolk, VA . This floral bed contained scores of Rudbeckia, along with a number of additional annual and perennial meadow flowers, some naturalized. The experiment was conducted from mid-September until mid-October in 2010 from ∼noon until 2:00 pm. Data were recorded and statistics performed as above.In a less cultivated, more natural setting at an urban sanctuary, a similar ‘cut-and-paste’ experiment was completed. R. hirta seed grown in the greenhouse were used for this study. This work was conducted on a grassy knoll, further away (∼25 m) from a smaller, mixed species floral garden at an urban zoo. This site allowed us to determine whether there were differences in the families recruited to this more monoculture-like urban habitat. Treatment one was another enhanced floral guide, here quite oversized, and comprising ∼90% of the petal surface. Treatment two was the ‘wild-type cut-and-paste control’ from above and treatment three was again a natural flower. Nine consecutive trials ran in full sun from ∼10:00 am to 2:00 pm per day in June of 2011. In addition to the Chi-square test and post-hoc tests described above here the cumulative  and mean visitation times were also recorded for all treatments. The variance in mean visitation times was large though homogenous  and these data were square root transformed prior to ANOVA.At this point we knew small floral guides were least desirable and we wanted to focus on whether large, uninterrupted guides were preferred. As well, to determine if our results held for more than one species, the native 1 = 2, d.f.2 = 29, P = 0.992) and these data were log10 transformed prior to ANOVA. Before we started this experiment, we conducted a food-color mark and recapture pilot study on Halictidae (n = 20) collected during a one-day trial. No marked visitors were identified the subsequent day but food-color marks remained on bees held for 24 hrs.This ‘oversized’ treatment experiment was repeated in September of 2011 for seven days. Here, insects were captured with mesh nets and removed after landing to ensure no repeat measures were included in the data set. The variance in mean visitation times was large though homogenous  were positioned ∼60 mm apart from one another in one long array, where treatment order repeated sequentially, and visitation monitored for a longer, single day time period. All of these experiments were conducted at the Virginia Zoo  in Norfolk, VA.This ‘oversized’ treatment experiment was repeated again in October of 2011, but here the design was a linear array. Thirty


Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles. Repetitive firing causes short-term depression (STD) at many synapsesshibire mutants led to a progressive decline in transmitter release during repetitive stimulation4Studies several decades ago revealed that intense stimulation slows the recovery of STD1Studies in the last 15 years have revealed that endocytosis can be rapid (1–2 s) or slow 10–30 s)69–30 s6910111314Slow endocytosis, mediated by a clathrin-dependent mechanism1619The RRP replenishment time course is often bi-exponential with a calcium/calmodulin-dependent rapid component of ~1 s or less2223242023Here, we provide a realistic three-pool model with all parameters experimentally measured, which shows the contribution of each step in exo- and endocytosis, and explains why the RRP replenishment is composed of rapid and slow components, and why the RRP replenishment could be slowed down by intense stimulation. We further applied this model to evaluate the contribution of endocytosis under prolonged action potential-like stimulation train. The current view of vesicle cycling may need to be modified to include the large recycling pool2-phosphocreatine, 0.3 GTP, 10 HEPES, 0.05 BAPTA . Measurements of the AMPA receptor-mediated EPSC were made by whole-cell patch at the postsynaptic principle neurons. The postsynaptic pipette (2.5–4 MΩ) solution contained (in mM): 125 K-gluconate, 20 KCl, 4 Mg-ATP, 10 Na2-phosphocreatine, 0.3 GTP, 10 HEPES, and 0.5 EGTA . The series resistance (<10 MΩ) was compensated by 90% (lag 10 μs). For recordings of the EPSC, kynurenic acid (1 mM) was added in the bath solution to relieve saturation and desensitisation of postsynaptic AMPA receptors. The holding potential was –80 mV for both presynaptic and postsynaptic recordings if not mentioned. Statistical analysis used a t test unless otherwise noted, and means are presented as ± SE. All the methods were carried out in accordance with the approved guidelines and all animal experimental protocols were approved by the Animal Care and Use Committee of Fudan University.Slice preparation was similar as described previously72620212, k-2) and between the IP and RRP . The kinetic scheme for these three pools is: With a large recycling pool ~46 times the size of RRP, we could not find any existing two-pool model that can fit the bi-exponential RRP replenishment with the rapid component being reduced by more intense stimulus201 and k-1, a slow rate constant for k2 and k-2, and a small IP size. The small IP could be depleted more by a more intense stimulus, explaining why the replenishmentrapid amplitude was decreased, instead of increased, after more intense stimulation. In the following, we performed quantitative calculations to determine the parameters  that fit our data.This model could explain the bi-exponential time course of replenishment by assuming a rapid rate constant for kFrom scheme 2,Based on 2 is a constant .The solution of this differential equation is: ∞ = A1 + A2. For simplicity, we normalised the RRP∞ to 1. Thus,Based on 1, k−1, k2, and k−2. These features are consistent with our experimental result that the RRP replenishment time course is bi-exponential, and that the time constants for rapid and slow RRP replenishment did not change significantly after 1–10 pulses of 20 ms depolarisation . Thus, RP∞ = 45 − IP∞. These calculations allow us to rewrite In the steady state (t = ∞), or resting conditions, 1 and t2 (the time constant for rapid and slow RRP replenishment) could be obtained by fitting experimental measurements after a single 20 ms depolarisation with ∞ was 1, and the normalised RP∞ = 45 - IP∞ (RRP∞ + IP∞ + RP∞ = 46). Thus, there are five unknown parameters, k1, k-1, k2, k-2, and IP∞ in four equations  from ∞ values and their corresponding rate constants , we numerically calculated the IP, RP, and RRP changes as a function of time after depletion of the RRP by a single 20 ms depolarisation. The numerical calculation was based on ∞ = 2.7, RP = 42.3, k1 = 0.8892, k-1 = 2.4008, k2 = 0.0093, and k-2 = 0.1546 (unit: sec-1).To solve these five parameters, we varied the IP1*[1 − exp(−t/τ1)] + A2*[1 − exp(−t/τ2)]) where A1 = 0.71, τ1 = 0.26 s, A2 = 0.29, τ2 = 9.5 s  decreased, whereas the slow component of replenishment (replenishmentslow) increased. Similarly, after 10 pulses of 20 ms depolarisation at 1 Hz, the RRP replenishment also slowed down . Our results contradict previous studies that suggested a calcium/calmodulin-dependent mechanism23After 10 pulses of 20 ms depolarisation at 10 Hz, which induced a capacitance jump of 1260 ± 72 fF (n = 11), the RRP replenishment could also fit a bi-exponential function with A = 7.8 s . Compare = 7.8 s , the raprapid amplitude was significantly reduced when calcium charge (QICa) was increased by ~8 times in rapid amplitude. Therefore, we proposed a new three-pool model to account for this phenomenon (see Methods for details).Our observation that the replenishment∞ + IP∞ + RP∞ = 46, where ∞ denotes the resting condition and all parameters are normalised to RRP∞), we found that the numerically calculated RRP replenishment time course after a 20 ms depolarisation best fit the observed data , k1 = 0.8892, k−1 = 2.4008, k2 = 0.0093, and k−2 = 0.1546 sec−1 , the RRP size can be derived as a function of time after depletion of the RRP. The analytical solution of the RRP  is a bi-ved data  when IP∞The above parameters were obtained by comparing the model with the observed RRP replenishment after a single 20 ms depolarisation . Third, the model-generated exocytosis during each time step matched well with the experimental results. As the model was derived from the RRP replenishment data, it would be supportive if the model could also predict exocytosis. For 10 depolarising pulses at 10 Hz, it is difficult to accurately measured the capacitance jump after each stimulusFirst, the model-predicted time course of the RRP replenishment after 10 pulses of 20 ms depolarisation at 10 Hz matched well with the observed time course  and the slow RP-IP kinetics  explained why replenishment was bi-exponential with rapid (τ1) and slow (τ2) time constants being controlled mostly by k1 and k−1, and k2 and k−2, respectively. Our numerical solution to τ1 and τ2 also further illustrated why the time constants in different stimulation protocols are roughly the same , we compared the influence of endocytosis in three circumstances: 1) endocytosed vesicles directly recycle within the RRP, 2) endocytosed vesicles enter the IP, and 3) the endocytosed vesicles enter the RP. Endocytosed vesicles did not directly enter the RRP because blocking endocytosis did not affect RRP replenishment . EndocytEndocytosis not affecting RRP replenishment raises doubts about the role of endocytosis. To determine the impact of endocytosis during repetitive firing, we mimicked the physiological firing frequency of 50 Hz by depleting the RRP by ~6–12% for each action potential in our model. We used 50 Hz because the calyx may fire spontaneously at a mean rate of ~50–60 Hz in vivo3436shibire mutants is consistent with our suggestion45In the absence of endocytosis, the model (scheme 2) predicted a gradual decline in release gradually to 0 . CompareBased on our previous finding that both rapid and slow endocytosis recycled vesicles to a large recycling pool instead of within the RRP or a small recycling pool, we proposed a model composed of three pools, a large RP ~42.3 times the size of RRP, a RRP and a small IP ~2.7 times the size of RRP in between13223541There are other explanations for the two components of RRP replenishment. Garcia-Perez et al. reported similar RRP replenishment at hippocampal synapses and provided “delayed depression” as an explanation, which lacked experimental verificationrapid with higher calcium influx during more intense stimulation  or the IP size is calcium/calmodulin-dependent.A previous study showing that blocking calmodulin function slowed down endocytosis and RRP replenishment suggested that slowing down of RRP replenishment under stronger stimulation might be caused by occlusion of the active zone by proteins participating in exocytosis6257Bi-exponential recovery from STD has been observed in many synapses13223541Although our three-pool model was proposed from RRP replenishment, it could also be used to accurately dissect the exocytosis at each time step during stimulation . FurtherJ.G. and L.X. designed research; J.-L.G., M.H. and X.-S.W. performed experiments; J.G., Z.-C.S. and L.X. built the model; J.-B. Z., W.W. and P.-T.Y helped with experiments; W.L. helped verify the model and L.X. supervised the project and wrote the paper.Supplementary information