Event-related potential (ERP) signatures of preparation to retrieve episodic memories have been identified in several studies. A common finding is relatively more positive-going ERP activity over right-frontal sites when people prepare for episodic rather than semantic retrieval. This activity has been linked to the process of retrieval mode – a retrieval set that ensures subsequent events are treated as cues for episodic retrieval. This experiment was designed to test one explanation for why this putative index of retrieval mode was not observed in two recent experiments. Towards this end, ERPs were recorded time-locked to different task-cues indicating which of two retrieval tasks participants should prepare to complete. Each task-cue was followed by a retrieval-cue that required a memory judgment. Departures from the designs of the two studies in which null ERP results were obtained were intra-trial timings and the order in which task cues were presented. Frequentist statistics revealed that ERPs elicited by the task-cues did index preparation to retrieve. The topographies of these activities, however, did not overlap markedly with that of the putative index of retrieval mode reported previously. Bayesian analyses, moreover, provided little compelling evidence for a signature of retrieval mode. These outcomes prompt consideration of how ERP sensitivities to preparatory retrieval processing should be characterized. Switch trials are those where the preparatory cue is different on the preceding trial. Repeat trials are those where the cue on the preceding trial matches that for the trial at hand . This seChanges in performance across switch and repeat trials – switch costs – have been documented extensively across many different kinds of task  differs from the 1200 ms RCI in the two studies reported by The argument for consideration of the predictability of task-cue sequences in the context of the key null outcomes is given added weight by the fact that unpredictable task-cue sequences have been employed in the majority of experiments in which neural activity indexing preparation to retrieve has been observed. In one notable exception, however, How might this timing difference between the experiment designs be important? In both studies there is the possibility that participants learned the task-cue sequence and started to prepare for the next trial in advance of the next task-cue. The extent of that preparation, however, is presumably more limited with a 500 ms RCI than with a 1200 ms RCI. If this is the case, then shorter RCIs will increase the likelihood of observing indices of preparation to retrieve when these are measured time-locked to a subsequent task-cue.This possibility still has the challenge of explaining why there were some divergences between the ERPs elicited by task cues in the first experiment At issue is the sensitivity of ERPs to processes that are engaged when people prepare to retrieve information from episodic memory. Failure to observe the putative ERP correlate of retrieval mode in this experiment would encourage re-consideration of its functional significance and prompt wider consideration of how best to characterize the array of processes that are in play when preparation for episodic retrieval is underway.22.1zd = 0.57, α = 0.05, 1 − β = 0.80, N = 22) and set at 24 to accommodate within experiment counterbalancing. A maximum sample size of 32 was again guided by power analyses: this N exceeds the largest sample size (N = 30) given from power analysis of the smallest previous effect size  were included. Data from a further three were excluded due to excessive EEG artefacts. The minimum sample size was based on power analyses for replication attempts of previous outcomes  . Two additional practice blocks, half the length of the other five study-test blocks, were formed and used before the task proper to familiarize participants with the experiment demands.At study, 24 of the objects were either presented inside or outside an abstract outline of a building see . They weAt test, the 24 objects from the preceding study phase were randomly intermixed with 24 unstudied objects. Each test object was preceded by one of two task-cues presented in the center of the screen. These cues indicated which task participants were to complete. An ‘X’ directed participants to prepare for the episodic task, where they were required to retrieve the prior study location of the object. An ‘O’ directed participants to prepare for the semantic task. This task required identification of the common location of the object depicted, regardless of the study phase. Each test block (for each task) was constructed such that it contained one four-trial run, two three-trial runs, five two-trial runs, and four one-trial runs. The order of these was randomized within each test block. This meant that within each block there were 12 switch trials and 12 repeat trials for each task. The data included in the analyses described below are for the first repeat only, for which there were 8 in each block.Whether an object appeared on a switch or repeat trial, its task status (episodic/semantic), and old/new status, were counterbalanced across participants. During the test phase, a three-way response was required in each task: episodic task: old-inside/old-outside/new, semantic: inside/outside/both. The inside/outside responses were made using the same fingers as at study, with the addition of the index finger of the other hand to indicate ‘new’ or ‘both’, for the episodic and the semantic task, respectively.2.3At study, a fixation asterisk was presented for 1000 ms, followed by an object (presented inside or outside the building frame outline) for 500 ms. The monitor was then blank until a response was made, and remained blank for 500 ms after each response before the next trial began.At test, the task-cue (‘X’ or ‘O’) appeared for 300 ms, followed by a fixation asterisk for 2000 ms. Following the ‘X’ cue, participants were to prepare to retrieve information about whether the object appeared inside or outside at study, or whether the object was new (response: inside/outside/new). Following the ‘O’ cue, participants were to prepare to identify where the object was most commonly found: inside or outside, or both inside and outside (response: inside/outside/both). An object was then presented in the center of the screen for 300 ms. The monitor was then blank until a response was made, and remained blank for a further 500 ms  before the next trial began .Participants were asked to respond as quickly and as accurately as possible. As in previous work , trials 2.4EEG was recorded from 25 silver/silver chloride electrodes housed in an elasticized cap. The arrangement of these was based on the international 10–20 system , encompaThe first trial in each test block was removed from analyses, as it is neither a switch nor a repeat trial. There were four conditions for which ERPs were extracted: those elicited by the episodic and semantic task-cues cues on switch and on repeat trials. On average, 84% of the available trials contributed to the ERP data for each participant. Mean trial numbers contributing to the ERPs (ranges in parentheses) were: episodic switch = 50 (31–56), episodic repeat = 34 (21–39), semantic switch = 49 (32–55), semantic repeat = 34 (23–40).33.1The likelihood of a correct inside/outside response at study was 97%. For the test data, Pr = p(hit) – p) were reliably above zero for both trial-types  = 26.80, p < 0.001, zd = 4.74, 99.9% CL; repeat Pr: 0.72, t(31) = 25.51, p < 0.001, zd = 4.51, 99.9% CL  = 1.89, p = 0.035, zd = 0.33, 63% CL). The probability of a false alarm was also higher on switch trials (t(31) = 2.71, p = 0.011, dz = 0.48, 68% CL).The probability of a correct old response was calculated as the summed probability of old words attracting a correct or incorrect location judgment. Old/new discrimination scores (discrimination index: t(31) = 11.84, p < 0.001, zd = 2.09, 98% CL) and repeat trials (t(31) = 15.40, p < 0.001, zd = 2.72, 99.7% CL). Accuracy was superior on repeat trials (t(31) = 3.56, p = 0.001, zd = 0.63, 74% CL). For the semantic task, the probability of classifying the item according to the modal rating given by the original raters was not statistically different across trial-types  = 16.81, p < 0.001, zd = 0.72, 77% CL). Main effects of old/new  = 30.04, p < 0.001, zd = 0.97, 83% CL) and task  = 15.12, p < 0.001, zd = 0.69, 75% CL) were moderated by an interaction between these factors  = 8.57, p = 0.006, p2η = 0.22). In both tasks reaction times are slower for ‘correct when old’ than for ‘correct when new’ judgments, and the interaction reflects the fact that the magnitude of this change is smaller for the semantic task (∼100 ms) than for the episodic task (∼200 ms).A 2 (old/new) × 2  × 2 (task) ANOVA was conducted on the mean per-participant reaction times (RTs) for correct responses see . As for 3.2The analysis strategy in this report followed that employed by F = 18.04, p < 0.001, p2η = 0.37). Separate ANOVAs were then carried out for switch and repeat trials. These revealed a task-cue by anterior-central by hemisphere interaction in both cases  = 10.03, p = 0.003, p2η = 0.24; repeat: F = 9.72, p = 0.004, p2η = 0. 24).The initial analysis revealed an interaction between task-cue, trial-type, anterior-central dimension, and hemisphere  = 8.94, p = 0.005, p2η = 0.22). Follow up ANOVAs were subsequently carried out for the anterior and central sites separately. For switch trials differences were reliable only at the anterior sites, where there was an interaction between cue-type and hemisphere  = 4.62, p = 0.04, p2η = 0. 13). For repeat trials differences were reliable only at central sites, where there was an interaction between task-cue-type and hemisphere  and in each case the time window of interest was 800–1900 ms after the presentation of the task-cues. When considered as a replication of the divergence obtained on switch trials in t-values and sample sizes for each of the key experiments.For switch trials, the outcomes in this experiment were set against those of For repeat trials, the outcomes obtained here were set against those in two separate experiments in which location judgments were required in an episodic task condition. The first experiment is that reported by The second experiment for the assessment on repeat trials is that reported by 4This experiment was designed to develop further an understanding of the conditions under which ERPs are sensitive to indices of preparatory retrieval processing. The focus was on the process of retrieval mode, which Tulving defined as a cognitive state, entry into which ensures that subsequent events – such as test items on a memory task – modulate episodic retrieval processing .The empirical starting point for this investigation was null results obtained in two recent experiments . In thosThe frequentist analyses described above provide some evidence that the ERPs elicited by the task-cues differed. On repeat trials there was greater relative negativity for neural activity elicited by episodic cues over left hemisphere central locations. On switch trials ERPs elicited by episodic cues were somewhat more positive-going than those elicited by semantic cues at right-frontal electrode locations and more negative-going at left hemisphere sites. This outcome was reflected in a reliable interaction between condition and hemisphere. No reliable differences according to condition were evident when analyses were conducted separately for each hemisphere. While the general frontal asymmetry on switch trials is broadly similar to that reported previously, the modulation is small, and the rather focal divergence around F8 that can be seen in How should these data be interpreted? In total, for four Bayesian analyses conducted over the data from three previously reported experiments, in each case restricted to electrode sites at which putative indices of retrieval mode have most commonly been observed, there was no strong evidence for the view that effects obtained previously were replicated. Moderate or strong evidence in favor of the null hypothesis was obtained in three of the four cases. In the earlier study by What other factors might explain the divergences across studies? In a recent report In light of these considerations, perhaps the most straightforward way to explain the set of outcomes described above is to consider other demands across the episodic tasks in this experiment, the two earlier highly similar ones , and in This observation raises the possibility that preparatory retrieval processing for memory judgments is distinct when the memoranda are either words or pictures, and that the functional significance of an effect linked until now to retrieval mode should be re-visited. The data from studies in which pictures were employed cannot be used to argue in favor of the view that retrieval mode itself is not a real psychological construct, but they do prompt consideration of how the right-frontal activity reported on several occasions is best conceptualized. Also of relevance to these considerations is the observation that visual inspection of the scalp distributions of the effects that have been linked in previous studies to retrieval mode reveals some divergences in their maxima as well as the extent to which they are lateralized to the right hemisphere . While dTurning to the behavioral data in this experiment, reaction time (RT) switch costs were observed. This outcome is consistent with the view that the processes necessary for accurate memory judgments are challenged by the requirement to switch between tasks frequently. There are data points consistent with the first of these two accounts. First, ERP old/new effects shown to be sensitive to the volume of recovered episodic contents are smaller on switch than repeat trials . Second,The findings do not, of course, rule out the possibility that preparing to retrieve influences retrieval processing in other ways as well, and any combination of these might be employed to explain the changes in response accuracy that were also observed in this experiment. Old/new discrimination was superior on repeat trials, as was the case in the two previous experiments reported by The final element of the behavioral data reported here is the superior location memory on repeat trials. This outcome has been observed in some studies and not in others, and the effects are commonly small cf: . These dIn conclusion, the ERP data reported here, when considered alongside data obtained in similar experiments, challenge the functional account offered previously for sustained right-frontally distributed activity that has been observed while people prepare for episodic retrieval. This activity has been linked to the process of retrieval mode. A key criterion for a neural index of retrieval mode is that it is engaged whenever episodic retrieval is required. The absence of strong evidence for this modulation in this experiment and in other similar ones suggests that the functional significance of this neural signature could be amended. It may well be an index of a retrieval orientation.Medical Research Council (MR/N01233X/1).AW was supported by a PhD studentship from the School of Psychology at Cardiff University, and is currently supported by a grant from the


Background: Venipuncture is described by children as one of the most painful and frightening medical procedures.Objective: To evaluate the effectiveness of Virtual Reality (VR) as a distraction technique to help control pain in children and adolescents undergoing venipuncture.Methods: Using a within-subjects design, fifteen patients  suffering from oncological or hematological diseases received one venipuncture with “No VR” and one venipuncture with “Yes VR” on two separate days (treatment order randomized). “Time spent thinking about pain”, “Pain Unpleasantness”, “Worst pain” the quality of VR experience, fun during the venipuncture and nausea were measured.Results: During VR, patients reported significant reductions in “Time spent thinking about pain,” “Pain unpleasantness,” and “Worst pain”. Patients also reported significantly more fun during VR, and reported a “Strong sense of going inside the computer-generated world” during VR. No side effects were reported.Conclusion: VR can be considered an effective distraction technique for children and adolescents’ pain management during venipuncture. Moreover, VR may elicit positive emotions, more than traditional distraction techniques. This could help patients cope with venipuncture in a non-stressful manner. Additional research and development is needed. For many children, venipuncture is one of the most frightening aspects of visiting a hospital . ExperieTraditional distraction techniques  are some of the most common psychological strategies for the reduction of procedural pain and anticipatory anxiety during venipuncture .n = 32 studies) and only two VR distraction studies were included in Birnie et al’s Cochrane review.Virtual Reality (VR) is showing promise as an innovative distraction technique for pain management among children undergoing medical procedures ,c. VR reThere is a growing interest in using VR for distraction among children and adolescents; however, to date most clinical studies on VR analgesia have included burn patients during physical therapy  or durinThe aim of the present study was to investigate VR effectiveness as a distraction technique for pain management in children and adolescents with onco-hematological diseases undergoing venipuncture. Based on the From February 2014 to July 2016, patients attending the Service of Pediatric Oncology and Hematological Diseases of an Italian Children’s hospital participated. Children and adolescents who needed to undergo venipuncture twice in a year, for intravenous placement during chemotherapy, transfusions, magnetic resonance or blood analysis were recruited. Patients were selected according to the following criteria based on the existent literature : childreSD = 2.64, see Table Seventeen patients met the inclusion criteria. However, one of them withdrew because he decided to use his own distraction technique and another patient withdrew because he didn’t want to use VR during the second venipuncture (the reason was not indicated). A total of 15 patients  or the experimental condition (“Yes VR”) (treatment order randomized). Patients underwent the second venipuncture using the distraction technique not used the first time. The “No VR” control condition consisted of non-medical conversation by the nurse who performed the venipuncture (standard of care). In the “Yes VR” condition, patients interacted with VR during venipuncture. Before the nurse arrived, patients had 5 min to learn how to use the VR system. The helmet and the earphones included in the VR system were worn at the arrival of the nurse for the procedure and removed after the procedure. In both conditions, during the venipuncture, the patient, a nurse, the patient’s parent/caregiver and the psychologist researcher were present. After the procedure, the nurse left the room and patients completed the self-report questionnaire (pain ratings). No reward was given to patients for participating.At the end of the procedure, patients filled out a brief self-report questionnaire aimed to evaluate the pain, the quality of the VR experience, nausea and fun. Patients responded by giving a 0–10 score on a horizontal Visual Analogue Scale VAS; . Pain, e2, one of the most frequently employed VR environments, specifically designed to promote distraction from procedural pain  vs. during “Yes VR” mean = 1.33 (SD = 1.05), p < 0.05, Cohen’s d = 0.62, moderate effect size; “Pain unpleasantness” during “No VR” mean = 3.27 (SD = 3.43) vs. during “Yes VR” mean = 0.93 (SD = 1.16), p < 0.01, Cohen’s d = 0.70 moderate effect size; “Worst pain” during “No VR” mean = 3.60 (SD = 3.00) vs. during “Yes VR” mean = 2.00 (SD = 1.20), p < 0.05, Cohen’s d = 0.51, moderate effect size.As reported in Table SD = 1.79), corresponding to “strong sense of going inside the computer generated world”, and a mean realism of VR objects score of 6.80 (SD = 2.37), corresponding to “Moderately real.”Patients distracted by VR reported a mean presence score of 7.93 (SD = 3.58) vs. “Yes VR” mean = 8.80, SD = 1.42; t(14) = −6.60, p < 0.0001, Cohen’s d = 1.71, large effect size. No significant differences emerged for nausea levels between the two conditions (p > 0.05 NS): no patient reported nausea during the interaction with VR. During the “Yes VR” condition the mean of the total time of the procedure was 3.09 min (SD = 1.81) vs. 4.45 (SD = 3.50) during the “No VR” condition. The pattern of results showed the venipuncture took less time during VR vs. during No VR; however, the differences were not significant .Patients rated “fun” during the venipuncture as “mildly fun” during No VR, vs. “pretty fun” during VR. A significant difference for fun levels emerged between the two conditions: during “No VR” mean = 2.93  are recommended.Future studies may explore whether personality aspects of the child, such as catastrophizing, fear of pain, as well as parents’ anxiety, and patients’ memory for previous painful procedures  influencThis study contributes to a growing literature that supports the use of immersive VR distraction for pain control. The current study evaluated the effectiveness of VR to control pain  and to promote fun during venipuncture in pediatric patients with cancer and blood diseases. Younger patients suffering from chronic diseases , who spend much time in hospital and need several painful and stressful medical procedures, could particularly benefit from this distraction. In the future, VR systems could also let patients have social interaction  and the All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Therefore, description of a new species, Nitzschia reskoi Ács, Duleba, C.E.Wetzel & Ector is proposed. We concluded that the increasing abundance of Nitzschia reskoi was a signal of the degradation of the intermittent saline wetlands.The spatial response of epiphytic diatom communities to environmental stress was studied in a moderately saline wetland area located in the plain of Danube-Tisza Interfluve, Hungary. The area is characterised by World War II bomb crater ponds and can be regarded as an excellent ecological model system where the dispersion of species is slightly limited by distance. To study the effect of environmental variables on the communities, canonical correspondence analysis was applied. Salinity, pH, total suspended solids, total phosphorous and depth proved to be significant environmental drivers in this analysis. The ecological status of the ponds was assessed with Ziemann’s halobity index, as the trophity-depending metric cannot be applied to these habitats . Ponds in “good” ecological status significantly differed from those appertaining to water quality category of “not-good” ecological status considering characteristic of natural astatic soda pans . The differences between epiphytic diatom communities inhabiting the ponds were detected using non-parametric multidimensional scaling. The samples formed three groups according to the types of ponds  based on the width of the macrophyte belt around them. Indicator species related to the ecological status of the ponds and diatom communities contributing to the separation of groups of ponds were identified. One of the indicator species differed from species already described. Light and scanning electron microscopy features and phylogenetic analyses based on three genes (18S and 28S rRNA genes,  Global warming affects the structure, function and stability of lake ecosystems throughout the world . Small, Saline lakes occur in every continent  and the +] and chloride [Cl-] ions) and the soda lakes . They can be also determined based on their hydrological cycles: perennial (or semiastatic) and astatic. Soda lakes are the most stable alkaline environments on Earth )n et al.  successfrbcL and 28S rRNA genes in comparison with other markers . They proposed a 748 bp long part at the 3’ end of rbcL (rbcL-3P) as a primary and D2-D3 region of the 28S rRNA gene as a secondary barcoding marker. The former marker could distinguish the most closely related ones of the studied taxa (these were Sellaphora isolates): the distance was 0.14% (2 bp) in the whole rbcL, 0.1–0.3% (1–2 bp) in rbcL-3P. Although rRNA gene D2/D3 region of the 28S rRNA gene could not resolve the species pair that rbcL-3P could, it could separate 96% of the studied species. The lowest distance was 0.3% (2 bp) in it. Based on the investigation of CCMP isolates the authors concluded that 1 bp difference in rbcL-3P and D2/D3 region of the 28S rRNA gene could indicate intraspecific variation. Our species showed much higher difference from the closely related species both in rbcL, rbcL-3P and 28S rRNA genes.Hamsher et al.  studied The sequence analysis results of the three markers suggest that the studied taxon from Apaj had differences from the published sequences of species of Bacillariaceae were high enough; therefore, it could be distinguished from them as a separate species.Nitzschia reskoi Ács, Duleba, C.E.Wetzel & EctorValve length 17–27 μm, width 2.4–3.7 μm, 19–30 striae in 10 μm, 9–12 fibulae in 10 μm. In SEM, externally the striae are uniseriate and are never doubled at the raphe keel. Areolae are irregular, circular, elliptical or sometimes missing within a stria. The first areola in the stria (near the raphe) has a thickened ring around them and the last one is elliptical. On the mantle, the striae are very short, comprising two areola, one of them is covered by hymen internally. Externally the distal raphe fissures hooked, internally terminating in small helictoglossae. The central nodule absent.Nitzschia frustulum, despite the lack of central nodule. Nitzschia reskoi prefers turbid, hypertrophic soda waters.The species is frequent in Hungarian saline lakes  and soda pans and presumably widespread in other inland saline lakes, but due to the uncertainity of its taxonomical position it has probably been identified occasionally as o13 on 8/05/2014.Holotype: MTA-ÖK-14/12, deposited at the Centre for Ecological Research, Danube Research Institute, Budapest, Hungary Type locality: Apaj, Hungary (47°7.403'N 19°8.187'E).Isotypes: HNHM-ALG-2261 deposited at the Hungarian Natural History Museum, Budapest, Hungary.Etymology: The name is in honor of Dr. Mária Reskóné Nagy, an Hungarian hydrobiologist who worked for many years on Lake Velence, an Hungarian soda lake.N. reskoi in Carpathian Basin are illustrated in N. reskoi in bomb crater ponds of Kiskunság National Park near Apaj (Nitzschia frustulum according to their micrographs) in the following aquatic systems: Bába-szék (indicated with 6 in Nitzschia frustulum: Albersee (1), Herrnsee (2), Kirchsee (3), Untersee (4), Zicklacke (5).Occurrences of ear Apaj , bomb crear Apaj  and LakeThe community-level response of epiphytic diatoms to natural environmental stress could be detected with non-parametric multidimensional scaling. The macrophyte belt determined the turbidity and transparency, therefore, we distinguish three different types: “turbid”, “transparent” and “transitional” ponds, which had different salinity, pH and TSS content that effected the composition of the epiphytic diatom communities. The “transparent” group was dominated by typical freshwater species mainly attaching with stalk, while in the “turbid” group adnate and motile halophylic diatoms prevailed. The “transitional” group was dominated by erected, adnate and motile forms as well.Epiphytic diatoms proved to be applicable in assessment of the ecological status of astatic soda ponds. Ponds in “good” and “not-good” status differing significantly in their environmental variables showed clear separation according to their epiphytic diatom community composition. This difference could be revealed in traits of diatoms: the “good” status could be characterised with higher proportion of motile or adnate, micro cell-sized diatoms with medium oxygen requirements and heterotrophic N-uptake strategy.Gomphonema species  and Achnanthidium minutissimum. Two Nitzschia species, N. liebetruthii and the newly described species as well as Navicula veneta and Psammodictyon constrictum were related to the “transitional” group. Dominance of Halamphora dominici, Nitzschia austriaca, N. supralitorea, Navicula wiesneri and N. radiosa Kützing characterized the “turbid” group.We identified indicator species of the three types of ponds according to the width of macrophyte belt. The “transparent” type was indicated by Halamphora dominici, Navicula wiesneri, Nitzschia austriaca, N. supralitorea indicated the “good” ecological status while A. minutissimum, Craticula buderi, G. angustatum, G. cf. micropus, G. clavatum, Nitzschia acidoclinata, N. commutata, N. palea and P. constrictum were related to the “not-good” status. The diatoms indicating the “not-good” ecological status are typical freshwater species. In most water types the increasing salt content indicates deterioration processes, in soda pans the disappearance of soda character is disadvantageous, so here we should choose a metric that indicates the changes of the salt content.Indicator species of “good” and “not-good” ecological status could be also determined. Nitzschia species was the most abundant in the ‘transitional’ group. Our results suggest that the increasing dominance of this species indicates deterioration of the soda character.We described a species new to science. This According to Reese et al. , a true Nitzschia austriaca and Halamphora dominici, which can reach strong dominance in the phytobenthos. The increasing abundance of Nitzschia reskoi seems to be a signal of losing sodic character of the intermittent saline wetlands. Our results may be used to design soda pan restoration plans in order for the soda pans to achieve the “good” ecological status corresponding to the requirements of the Water Framework Directive.These saline bomb crater ponds are good inference models for intermittent soda pans of the Carpathian Basin because they share many common features  with the Central European astatic soda pans. To define reference conditions and select their variations due to natural stress is a great challenge of ecological status assessment. On the studied area, no human intervention has occurred since the World War II bombing, because it belongs to a national park. Therefore, primarily the ponds belonging to the "turbid" group can be model sites for the shallow, astatic soda lakes. Their indicators are mainly the species S1 Table(DOCX)Click here for additional data file.S2 TableH = high, G = good, M = moderate, P = poor, B = bad. The ponds were grouped according to the macrophyte belt: 1 = “transparent”, 2 = “transitional”, 3 = “turbid”. EQR: ecological quality ratio.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 FigTypical pictures of bomb crater ponds of “transparent” (A), “transitional” (B) and “turbid” (C) groups.(TIF)Click here for additional data file.


Lactoperoxidase , as a natural antibacterial agent, is a peroxidase enzyme secreted from salivary, mammary, and other mucosal glands. In the present study, the in vitro inhibitory effects of some secondary sulfonamide derivatives (4a–8h) were examined against LPO. The obtained results reveal that secondary sulfonamide derivatives (4a–8h) are effective LPO inhibitors. The Ki values of secondary sulfonamide derivatives (4a–8h) were found in the range of 1.096 × 10−3 to 1203.83 µM against LPO. However, the most effective inhibition was found for N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a), with Ki values of 1.096 × 10−3 ± 0.471 × 10−3 µM as non-competitive inhibition.Secondary sulfonamides ( Sulfonamides constitute a privileged class among pharmacological agents by possessing properties including carbonic anhydrase enzyme (CA) inhibition, as well as diuretic, hypoglycemic, anticancer, antibacterial, antiviral, and metalloprotease inhibitory effects. Although many years passed since their first discovery in the 1930s as a chemotherapeutic agent for antibacterial properties they are receiving increasing attention due to their newly-discovered pharmacological properties ,3,4,5. DIt was also reported that sulfonamides had a number of interesting functionalities. Some example can be given for secondary and tertiary and sulfonylureas represent particularly important structural motifs in several classes of drugs. Sulfonamides possessed some biological activities as antibiotics (sulfhamethoxazole), PDE5 inhibitors for the treatment of erectile dysfunction (sildenafil), protease inhibitor for treatment of HIV (darunavir), sulfonylureas for treatment of diabetes mellitus (glibenclamide), hepatitis C anti-viral RNA polymerase inhibitors, and non-steroidal anti-inflammatory COX-2 inhibitors (oxicam class) .Because secondary sulfonamides have been reported to be effective agents, in this study, new designs of secondary sulfonamide drugs containing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole moieties were utilized to obtain acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide and trihydroxybenzamide sulfonamide derivatives to achieve the synergistic effects of sulfonamides and polyphenols in one molecule. Since acetyl protection of hydroxyl groups is a well-known application in pharmacology to enhance oral bioavailability and to increase therapeutic concentration of the drug in the bloodstream, both acetylated and non-acetylated forms of compounds were examined together.2O2), and thiocyanate (SCN−). H2O2 is produced by a number of microorganisms such as lactobacilli, Lactococcus and streptococci through enzymatic oxidation of some biomolecules including ascorbic acid and glucose  plot was drawn. For the determination of the inhibition constant (Ki) values, three different secondary sulfonamide derivative (4a–8h) concentrations were used. Additionally, ABTS was used as a substrate at five different concentrations. Lineweaver–Burk plots (1/V-1/[ABTS]) were obtained for secondary sulfonamide; the Ki and the inhibition type were calculated from these plots , which showed parallel lines for different concentrations of secondary sulfonamide derivatives (4a–8h). The lines do not intersect at a common point as shown in 4a–8h) inhibition effects against LPO are commonly non-competitive with respect to the ABTS substrates. In this assay, it was suggested that the secondary sulfonamides (4a–8h) do not directly react with LPO. However, any variation in the concentration of ABTS does not affect the reactivity of secondary sulfonamides (4a–8h). This is also reflected in the non-competitive nature of the inhibition by the compounds with respect to ABTS. The inhibition data of secondary sulfonamides (4a–8h) reported here are summarized in 4a–8h) is dependent on the positioning of the inhibitor in the active site; i.e., the distance between the atoms in the secondary sulfonamide derivatives (4a–8h) and active site amino acids.Although sulfonamide derivatives are used in therapies, there has been no reported LPO activity for these synthesized compounds (of 75.6% . The pur for LPO . For sec4a–8h) exhibited effective inhibitory activity against LPO, one of the prominent enzymes in milk, with a Ki values in the range of 1.096 × 10−3 ± 0.471 × 10−3–1203.83 ± 616.78 µM (N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a) demonstrated the most powerful LPO inhibition effect with low nanomolar Ki value (1.096 nM). On the other hand, among the synthesized secondary sulfonamides (4a–8h) N-(sulfadiazine)-p-acetoxy-benzamide , N-(sulfamethazine)-p-acetoxybenzamide , N-(sulfisoxazole) p-acetoxybenzamide , N-(sulfamethizole)-p-acetoxybenzamide , N-(sulfanilamide)-p-hydroxybenzamide  and N-(sulfathiazole)-3,4,5-trihydroxybenzamide , showed effective inhibition profiles against bovine milk LPO. Additionally, in general, the acetylated sulfonamides are better inhibitors than the non-acetylated sulfonamides, although the acetylated acids are poorer inhibitors than the non-acetylated acids.All synthesized secondary sulfonamides derivatives  to the antibacterial hypothiocyanite (OSCN−). Secondary sulfonamides (4a–8h) have shown micromolar to nanomolar inhibition against LPO that is vital activity for the innate immune system because of removing bacteria from milk and mucosal secretions. If LPO activity is reduced, this means that the immune system is weakened. This is particularly undesirable, since this affects the immune system of infants that used sulfonamides as a drug.Secondary sulfonamides incorporating acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide and trihydroxybenzamide–all possessing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole groups–were synthesized, and biological activities were evaluated. Milk is a vital liquid secreted from the mammary glands of females of all mammal species. So, the inhibition of the LPO is very important in terms of the LPO system, which catalyses the oxidation of several different reactions by H


Most reported that professional and semi-professional athletes’ dietary intakes met or exceeded recommendations during training and competition for protein and/or fat, but not energy and carbohydrate. Limitations in articles include small sample sizes, heterogeneity of data and existence of underreporting. Conclusions: this review highlights the need for sport-specific dietary recommendations that focus on energy and carbohydrate intake. Further exploration of factors influencing athletes’ dietary intakes including why athletes’ dietary intakes do not meet energy and/or carbohydrate recommendations is required.Background: to develop sport-specific and effective dietary advice, it is important to understand the dietary intakes of team sport athletes. This systematic literature review aims to (1) assess the dietary intakes of professional and semi-professional team sport athletes and (2) to identify priority areas for dietetic intervention. Methods: an extensive search of MEDLINE, Sports DISCUS, CINAHL, Web of Science, and Scopus databases in April–May 2018 was conducted and identified 646 studies. Included studies recruited team sport, competitive  athletes over the age of 18 years. An assessment of dietary intake in studies was required and due to the variability of data  a meta-analysis was not undertaken. Two independent authors extracted data using a standardised process. Results: 21 ( Team sports can be defined as field- and court-based sports with intermittent and high-intensity game patterns . Match pDietitians must consider a range of sport-specific factors including the rules, arena size, timing of competition, frequency of matches and length of seasons  when assessing an athlete’s nutrition requirements and goals. Additionally, the physique characteristics and position-specific tasks of the sport will further influence the nutritional requirements of athletes. For example, the sport of rugby union will require forwards to be heavier and stronger in comparison to backs who need to be leaner and faster . Due to Recommendations that support athletes to consume sufficient energy and the correct balance of macronutrients and micronutrients, with appropriate timing to enhance performance and recovery, will enable athletes to train and perform optimally . An earlIn the past decade, thousands of new research papers have been published in sports nutrition and 17 new consensus statements and recommendation papers have been released by authoritative organisations such as the IOC, ACSM, ISSN ,20,21,22All methods and search strategies were aligned with Preferred Reporting Items for Systematic Review (PRISMA) guidelines. This review was registered with International Prospective Register of Systematic Reviews (registration number: CRD42018105168) ,41. A PIFive electronic databases including MEDLINE , Sports DISCUS, CINAHL , Web of Science (WOS), and Scopus (World’s largest abstract and citation database of peer-reviewed literature) were searched to investigate the dietary intake of professional and semi-professional team sport athletes by one researcher (SJ). The timeframe designated for the search included studies published from 2011 to present  [−1·day−1), protein (g and g·kg−1·day−1), fat , calcium (mg/day) and iron (mg/day). Studies that reported dietary habits, dietary knowledge, attitudes, and education strategies where dietary intake was not quantified, were excluded from the review.All original research  conducted in adult team sport athletes  and published since 2011 was considered for inclusion . RandomiAll studies were screened based on title and abstract by main author (SJ). Articles deemed eligible for full text review were screened against inclusion and exclusion criteria  by two a−1·day−1), protein intake , fat intake , calcium intake  and iron intake (mg). Where total energy intake was reported in calories, this was converted to MJ to enable comparison across studies.Dietary intake data were extracted from the included studies by two authors (SJ and GB). Data presented in The quality of studies was examined by two authors (SJ and GB) using the Academy of Nutrition and Dietetics Quality Criteria Checklist from the Academy of Nutrition and Dietetics Evidence Analysis Manual . The quaThe original search retrieved 646 studies that fit the search criteria with an additional 15 studies identified by hand searches . After dn = 255) [n = 81) [n = 34) [n = 30) [n = 26) [n = 25) [n = 19) [n = 14) [n = 11) [n = 10) [n = 6) [n = 210) [n = 139) [n = 70) [n = 25) [n = 17) [n = 15) [n = 14) [n = 11) [n = 10) [The majority of studies included in this review included team sport athletes competing professionally and semi-professionally in Australia n = 5 [25,26,n = 255) ,43 and S[n = 81) ,29,30,36[n = 34) ,27, Engl[n = 30) ,34, Amer[n = 26) ,38, Cana[n = 25) , Brazil [n = 19) , Netherl[n = 14) , South A[n = 11) , United  [n = 6) ,37. The  [n = 6) ,34,36,37 [n = 6) ,35,38,39n = 210) ,34,37, f[n = 70) ,35, ice-[n = 15) , handbaln = 14), with the remaining studies using pre-post-test [The majority of studies included in this review used a cross-sectional study design  did not report on adequacy of energy intake  [All studies included assessed the energy intake of team sport athletes. The majority of authors assessed the energy intake of athletes against recommendations advocated by IOC, ACSM, ISSN and sports specific research ,53,54,55not met) ,30,31,34n = 10) and ice hockey (n = 25) athletes on match days found that energy intake did not meet increased requirements for the fueling and recovery required on match days [n = 14) and football athletes (n = 6), found that energy intake was greater on match days in comparison to training days [n = 6) had a greater absolute and relative energy intake on match days in comparison to training days.The mean energy intake reported in this review ranged from 9.1–16.6 MJ/day and 7.3–9.8 MJ/day for males and females respectively. Seven studies included comparison data of dietary intake on training days and match days ,31,35,39ckey n = 5 athleteing days ,27. In ping days  additionn = 175), Australian football (n = 139), rugby union (n = 88), volleyball (n = 11) and rugby league (n = 10) consumed diets that consistently did not meet carbohydrate recommendations [−1·day−1 . One study that explored the dietary intakes of male professional football players found that carbohydrate intakes consumed by athletes were closer to meeting recommendations for tactical or skill based sports (3–5 g·kg−1·day−1) [Overall, the macronutrient composition of the diets of team sport athletes was inadequate to meet the fuel, recovery and performance demands of their sports. All but one study assessed the dietary intake of carbohydrate and protein  and all all n = 19, rugby ague n =  consumedndations ,37,38,431·day−1) ,25.−1·day−1 [−1·day−1 of protein per day [n = 35) and football athletes (n = 25) found that while overall total fat intake exceeded recommendations, polyunsaturated fat intake fell below recommended intakes of 10% of total energy [Conversely, the majority of studies found that dietary intake of protein ,37,43,56−1·day−1 ,33,35,43 per day ,38,43. N per day ,35,37,39l energy ,35,37. Setes n = 5 found tl energy ,32,35,37p = < 0.05; 6.4 g·kg−1·day−1 and 4.2 g·kg−1·day−1 for match and training days respectively). In comparison, research by Bettonviel et al. [−1·day−1) and one day post-match (4.5 ± 1.0 g·kg−1·day−1); however, protein intakes on match day and post-match were adequate (2.0 ± 0.4 and 1.6 ± 0.3 g·kg−1·day−1 respectively).Three of seven studies that assessed intake on competition days found that carbohydrate consumed in preparation for match day, during the match and during the recovery period post game, did not meet recommended intakes for competitions ,31,39. Ol et al.  found thSix studies reported iron and/or calcium intakes ,36,37,39In order for athletes to optimise training and performance, they need to consume sufficient energy for the work required and to support physiological adaptions ,13,57. AWhile not identified as an influencing factor previously , three s−1·day−1 (3.5 g·kg−1·day−1 and 3.4 g·kg−1·day−1 for forwards and backs respectively). Although these intakes did not align with recommendations, Bradley et al. [fueling for the work required” is commonly being used by many sports nutrition professionals working in professional sport [Carbohydrate intake is important for optimising performance and recovery. Team sports have varied training and physiological demands, therefore advice must be tailored to match the training demands as well as the demands of specific positions within the sport. Team sport athletes included in this review continue to consume diets that do not align with carbohydrate recommendations, intakes on average insufficient when compared to recommendations ,11,12,52y et al.  suggesteal sport . The conal sport  found th−1·day−1 of high-quality protein [−1·day−1) may be appropriate for resistance trained athletes who are consuming hypocaloric diets with the aim to maintain lean muscle tissue while reducing fat mass [−1·day−1) exceeded protein recommendations, since athletes were manipulating carbohydrate intakes during the preseason training week, protein intakes may have been appropriate at the time to optimise body composition. Given the context of the time of season this study was undertaken  and the ability for athletes to optimise training adaptions and changes to body composition without any detriment to competition performance, these intakes may be acceptable for this athletic cohort. However, in contrast, research by Potgieter et al. [−1·day−1) may be suited in times where muscle hypertrophy is required ; however, in-season where athletes are required to meet training, competition and recovery demands, protein intakes should align with recommendations and not be increased at the expense of carbohydrate intake. Similarly, research by Mackenzie et al. [Protein is important for muscle protein synthesis, supports recovery processes, promotes satiety and can aid the maintenance of body composition. It also has many other important roles in the body as enzymes, hormones, transporters and antibodies. Athletes with insufficient protein intakes have an increased risk of muscle wasting, illness and injury. ISSN recommendations state that to maintain protein balance athletes should consume 1.4–2.0 g·kg protein . In this protein ,36,38,43fat mass ,60. Thisfat mass  who founr et al.  reportedr et al.  suggestse et al.  on rugbye et al. . In addiFour of the studies that reported athletes’ diets exceeding recommended fat intakes  included the dietary intakes of athletes competing in strength and power sports such as rugby union 2), rugby league 1) and American football (1) , rugby l,32,35. C and AmerAlthough most team sport athletes included in this review consumed diets that were in line with or exceeded recommendations for dietary fat intake, the composition of saturated to unsaturated fats  was suboptimal . This is supported by Hidalgo et al.  who founThis review highlights the role of dietitians in providing long-term dietary strategies to increase lean muscle and total body mass, in a manner that does not adversely affect performance and/or lipid profile, which may be evident in short term high energy and high fat diets. Greater education regarding the long-term implications of intakes that are in excess of total fat and saturated fat is required in team sport environments.−1·day−1) for match days; however, in comparison these athletes exceeded recommended protein intakes. Additionally, research by Potgieter et al. [Due to the elevated requirements for stored glycogen and glycogen resynthesis during training and competition, athletes are recommended to undertake aggressive carbohydrate feeding prior to these periods. Inconsistencies between energy and carbohydrate intakes on training and match days were observed in team sport athletes. In particular research by Bettonviel et al.  found thr et al.  found thr et al.  found thr et al. ,62. Grear et al. . In thesr et al. .Studies in this systematic review included small numbers of participants and may not be generalisable to team sport disciplines more broadly. In addition, the heterogeneity of the included studies led to an inability to compare results across all studies and as a result a meta-analysis of data was not possible. Underreporting is an important consideration when assessing dietary intake; however, suboptimal intakes should not be attributed solely to underreporting and dietary assessment should encompass a range of influencing factors  . Many stThis systematic review found that despite the publication of high-quality research studies, expert consensus statements and recognition of the consequences of inadequate intakes, team sport athletes’ total energy and carbohydrate intakes did not meet sports nutrition recommendations  for energy and carbohydrate. In contrast, many athletes met or exceeded recommendations for protein and/or fat. Further research into the development of sport-specific recommendations for energy and macronutrients in particular carbohydrate would be beneficial to further optimise distribution throughout a training week. Furthermore, nutrition in team sport environments requires a knowledge base of the physiological demands of training and competition, and therefore sports dietitians should work collaboratively with sports science teams when tailoring nutrition advice to meet energy and macronutrient needs. Future research is required to explore the factors that influence athletes’ dietary intakes.


Dimocarpus longan Lour.) is an important fruit tree in the subtropical regions of Southeast Asia and Australia. Among the factors affecting D. longan fruit yield, the difficulty and instability of blossoming is one of the most challenging issues. Perpetual flowering (PF) is a crucial trait for fruit trees and is directly linked to production potential. Therefore, studying the molecular regulatory mechanism of longan PF traits is crucial for understanding and solving problems related to flowering. In this study, comparative transcriptome analysis was performed using two longan cultivars that display opposite flowering phenotypes during floral induction.Longan (COL), two-component response regulator-like (APRRs), gigantea (GI), and early flowering (EFL). In addition, the leafy (LFY) gene, which is the central floral meristem identity gene, may inhibit PF formation in ‘SJ’.We obtained 853.72 M clean reads comprising 125.08 Gb. After comparing these data with the longan genome, 27,266 known genes and 1913 new genes were detected. Significant differences in gene expression were observed between the two genotypes, with 6150 and 6202 differentially expressed genes (DEGs) for ‘SJ’ and ‘SX’, respectively. The transcriptional landscape of floral transition at the early stage was very different in these two longan genotypes with respect to key hormones, circadian rhythm, sugar metabolism, and transcription factors. Almost all flowering-related DEGs identified are involved in photoperiod and circadian clock pathways, such as CONSTANS-like  longan genotypes and may benefit studies on PF trait mechanisms of evergreen fruit trees.The online version of this article (10.1186/s12864-019-5461-3) contains supplementary material, which is available to authorized users. Dimocarpus longan Lour.) is an important subtropical fruit tree that is widely grown in several subtropical and tropical countries [D. longan requires favorable conditions such as a period of low temperature , suitable salinity and dry conditions. To obtain a stable high yield, longan flowering in the off-season is achieved through chemical treatment with potassium chlorate (KClO3) [Longan  , 5, and Arabidopsis and other model plants, the molecular mechanisms of flowering have been well established. There are at least five major flowering pathways in Arabidopsis, including photoperiod, autonomous, vernalization, gibberellin (GA), and aging pathways [FT), flowering locus C (FLC), and CONSTANS (CO) [Arabidopsis LFY in poplar (Populus spp.) resulted in early flowering in the transgenic lines [FT1 and FT2, whereas the first flowers on wild-type trees were obtained after 5–10 years [Chrysanthemum FTL1 in Arabidopsis caused varying degrees of early flowering [FT1 and FT2 have been ectopically expressed in Arabidopsis, whereby DlFT1-and DlFT2-overexpressing lines showed early flowering and late flowering phenotypes, respectively. Interestingly, transgenic Arabidopsis lines overexpressing longan apetala1 (AP1) displayed a range of flowering time phenotypes [Arabidopsis consistently fail to affect blossoming in trees. For example, overexpressed MADS1 and CO, LFY, AP1, and agamous-like20 (AGL20) in poplar, the transgenic lines showed very rare and early floral onset or no flowers, suggesting the presence of different flowering regulatory mechanisms in perennials [As an important developmental process in the plant life cycle, flowering is directly linked to production, regardless of when seeds or fruits are harvested , and thupathways , that acANS (CO) . HoweverANS (CO) . For exaic lines , and flo10 years , 12. In lowering . Moreoveenotypes . Nonetherennials .PEP1), an ortholog of the floral repressor FLC, controls the PF trait in Arabidopsis [TFL1) [FaPFRU locus, a nonortholog of TFL1, controls the PF trait in some strawberry cultivars [Because it extends the production period, PF is a crucial trait for fruit trees , and thebidopsis , whereass [TFL1) , 18. Recultivars , 20. Regultivars , 5, the D. longan ‘SJ’ and ‘SX’ (nine-year-old trees), which display opposite flowering phenotypes, were grown at an experimental orchard of the South Subtropical Crops Research Institute of the Chinese Academy of Tropical Agricultural Science in Zhanjiang, China . ‘SX’, one of the main varieties in China originating from Guangxi Province, exhibits SF traits, and floral bud induction requires a period of low temperature [perature . Previouperature . Three dTotal RNA was obtained using a quick RNA Isolation Kit  according to the manufacturer’s instructions, and contaminant DNA was removed. The concentration and quality of the RNA were verified using an Agilent 2100 Bioanalyzer . Equal amounts of total RNA extracted from the three replicate plants at each flowering time comprised the cDNA library. Eighteen cDNA libraries (2 cultivars × 3 flowering times × 3 replicates) were constructed and sequenced using the Illumina HiSeq™ 2000 platform . Before assembly, adaptor sequences were removed from the raw reads. To obtain more reliable results, low-quality reads with over 50% bases were removed from each dataset. Those with quality scores of 5 or lower and/or over 10% bases unknown (N bases) were also removed. High-quality clean reads from 18 samples were mapped to the longan genome database  using SOp values were adjusted using the Benjamini and Hochberg approach for controlling the false discovery rate (FDR). Genes with a minimal 2-fold difference in expression (|log2 Ratio| ≥ 1) and an adjusted p-value < 0.05 were considered differentially expressed [p-value < 0.05 were considered significantly different from the reference set for each genotype.The level of unigene expression was normalized by calculating the reads per kilobase of exon model per million mapped reads (RPKM), as estimated using RSEM v1.2.15 . To detexpressed . To evalxpressed . The clup-value (p.adjust) ≤ 0.05 were considered significantly enriched. DEGs were mapped to terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using BLASTX software [To verify biological significance, all DEGs were subjected to Gene Ontology (GO) enrichment analysis using Blast2GO (version 2.8)  and WEGOsoftware . Pathway2-transformed, and heat maps with hierarchical clustering were generated using the software Mev4.9.0 [Transcription factors (TFs) were obtained from a previous study , and MapMev4.9.0 .actin (Dlo_028674), which was used in our previous study, was selected as the reference gene [-∆∆Ct method [Thirty-eight DEGs were selected to confirm the transcriptomic data through qRT-PCR analysis. Gene-specific primers were designed using Primer 3 software  Sequence Read Archive (SRS2241241–SRS2241258). After filtering adaptor sequences and removing low-quality tags, 853.72 M clean reads were obtained, with 41,542,306–57,566,164 clean reads generated from the 18 samples and 125.08 Gb of sequence. The Q30 percentage (sequences with a sequencing error rate lower than 0.1%) was greater than 93%, and the average GC content was 44.70%. Among the total clean reads, approximately 32,322,773 (77.81%)–47,947,255 (83.29%) matched perfectly to the longan genome , 3878 DEGs in T2 (SXT2-vs-SJT2) and 4919 DEGs in T3 (SXT3-vs-SJT3)  are shown in Additional file DEGs were first identified through comparisons of the RPKM values for each gene of ‘SJ’ and ‘SX’ at different floral induction stages  with |logq-value = 9.64 × 10− 3, 51 genes) and “plant hormone signal transduction” . For the DEG comparison between ‘SJ’ and ‘SX’ at T2, pathways of “plant-pathogen interaction” , “plant hormone signal transduction” , and “circadian rhythm-plant”  were significantly enriched. Finally, “plant hormone signal transduction”  and “plant-pathogen interaction”  were significantly enriched DEGs between ‘SJ’ and ‘SX’ at the T3 stage.In the SXT1-vs-SJT1, SXT2-vs-SJT2, and SXT3-vs-SJT3 comparisons, 813, 167, and 402 DEGs were mapped to 118, 72, and 98 KEGG pathways, respectively. Ten pathways were significantly enriched the SXT1-vs-SJT1 comparison  are mainly related to genes involved in plant basal metabolic processes; DEGs between ‘SJ’ and ‘SX’ at T2 and T3 are mainly involved in signal transduction and environmental adaptation. Interestingly, genes involved in circadian rhythm were only significantly enriched in the T2 stage, the emergence of the floral primordia, which is the signal for flower initiation.Gene expression over the developmental course of flower induction was comparatively examined , revealing that 6150 and 6202 DEGs were significantly expressed in ‘SJ’ and ‘SX’, respectively Fig. . Moreovep < 0.05  , “photosynthesis–antenna proteins” , and “plant-pathogen interaction” . Expression of the DEGs in profile 0 was decreased during the entire stage. Profile 1 showed more significantly enriched GO terms than did profile 0, and most of these 140 significantly enriched GO terms are related to “binging”, “catalytic activity”, “stress response”, and “regulation of biological process”. Moreover, genes involved in “limonene and pinene degradation” , “stilbenoid, diarylheptanoid and gingerol biosynthesis” , “plant-pathogen interaction” , “benzoxazinoid biosynthesis” , and “plant hormone signal transduction”  were significantly enriched. Gene expression decreased through T1 to T2 and remained low during the subsequent stage. In profile 6, there were 96 significantly enriched GO terms, with most DEGs being involved in “cellular biosynthetic”, “macromolecule biosynthetic”, “intracellular component biosynthetic” and other processes associated with cell proliferation and differentiation. The significantly enriched pathways of this profile were found to be “starch and sucrose metabolism” , “ascorbate and aldarate metabolism” , “phenylpropanoid biosynthesis” , “pentose and glucuronate interconversions” , “plant hormone signal transduction” , “DNA replication” , “carotenoid biosynthesis” , and “flavonoid biosynthesis” . The expression level of these genes in profile 6 peaked at T2 and remained high during T2 to T3. However, only two GO terms (“sequence-specific DNA binding transcription factor activity” and “nucleic acid binding transcription factor activity”) and one pathway  were significantly enriched in profile 7, and gene expression in this profile increased during the entire stage.To evaluate the expression profiles, trend analyses were performed using STEM software, and the DEGs in ‘SJ’ and ‘SX’ clustered into 8 profiles. In ‘SJ’, 4 profiles had a significance of ly) Fig. . Profileq-value < 0.05 , “plant-pathogen interaction” , “stilbenoid, diarylheptanoid and gingerol biosynthesis” , “plant hormone signal transduction” , “limonene and pinene degradation” , and “diterpenoid biosynthesis”  were overrepresented. DEGs involved in “photosynthesis” and “regulation of biosynthetic process” were significantly enriched in profile 3, as were pathways of “diterpenoid biosynthesis” , “photosynthesis” , “circadian rhythm”  and “plant hormone signal transduction” ; gene expression remained at a high level at T1 to T2 and decreased during the subsequent stage. DEGs in profile 4 remained at a low level from T1 to T2 and increased during the subsequent stage. Overrepresentation of “DNA replication” , “stilbenoid, diarylheptanoid and gingerol biosynthesis” , “limonene and pinene degradation” , “alpha-linolenic acid metabolism” , “pyrimidine metabolism” , “phenylpropanoid biosynthesis” , “base excision repair” , “plant hormone signal transduction” , “linoleic acid metabolism” , and “cysteine and methionine metabolism”  were enriched in this profile. In turn, profile 7 contained more DEGs significantly enriched in “catalytic activity”, “single-organism process” and cell wall association function categories, and significantly enriched pathways were “carotenoid biosynthesis” , “ascorbate and aldarate metabolism” , “pyruvate metabolism” , and “riboflavin metabolism”  (Tables S8 and S9).In ‘SX’, 6202 DEGs clustered into 8 profiles. We also found 4 profiles with a significant beta-glucosidase 40 (Dlo_026115.1), sucrose synthase (Dlo_019926.1), and endoglucanase 17 (Dlo_023528.1) , whereas the major transcriptional regulation of ‘SX’ occurred at the period of floral organ formation (T2 to T3 transition). DEGs in starch and sucrose metabolism pathways were only significantly enriched in profile 6 of ‘SJ’, with increased expression at T1 to T2 for Dlo_02352.1 , beta-glucosidase, fructokinase, and endoglucanase , TPS , starch synthase, PYG, alpha-glucosidase, sucrose-phosphate synthase, HK (hexokinase), and pgm (phosphoglucomutase). However, only 4 genes  and 3 genes  were significantly upregulated or downregulated at the same stage in ‘SX’. There were 13 and 2 common genes significantly upregulated or downregulated, respectively, in these two cultivars. No genes in ‘SJ’ were significantly up- or downregulated during the T2 to T3 transition. Although 23 and 9 genes were significantly upregulated and downregulated at the same stage in ‘SX’, 4 genes common to these two cultivars were significantly upregulated.As the starch and sucrose metabolism pathway was only significantly enriched in profile 6 in ‘SJ’ and the SXT1-vs-SJT1 comparison, the 97 DEGs , five auxin-responsive genes, two indole-3-acetic acid-amido synthetase (GH3) genes, and six SAUR family genes, showed upregulation, and one SAUR32-like gene (Dlo_023207.1) and one auxin-responsive gene (Dlo_021779.1) showed downregulation; 16 genes related to the CK pathway were upregulated, and 3 genes were downregulated, including AHK and ARR-A. Moreover, 11 genes related to the GA pathway were upregulated, including 7 gibberellin receptor GID1 and 4 DELLA genes, and 3 GID1 and 2 DELLA genes were downregulated. Two PP2C and 2 PYL genes associated with the ABA pathway were upregulated, and 2 PP2C, 1 SRK2, and 1 ABF were downregulated. In addition, 33 and 7 DEGs involved in the brassinosteroid signaling pathway exhibited upregulation or downregulation, including BRI1, BSK, BRZ1/2 and CYCD3; one CTR1 (Dlo_013291.1) and one ERF1 (Dlo_022309.1) related to the ET pathway showed upregulation, and one EIN3 (Dlo_030832.1) and one ERF1 (Dlo_008317.1) displayed downregulation. Eight and two DEGs related to the JA pathway were upregulated or downregulated, respectively, during T1 to T2. Three TGA TFs related to the SA pathway displayed downregulation in ‘SJ’ during T1 to T2. In ‘SJ’, only AUX1 (XLOC_010561) and PIF3 (Dlo_027777) were upregulated or downregulated during T2 to T3. Compared to ‘SJ’, most DEGs related to hormone biosynthesis and signaling pathways in ‘SX’ were enriched in the late floral induction stage: 43 DEGs were upregulated or downregulated during T1 to T2. In contrast, 92 DEGs displayed upregulation or downregulation in ‘SX’ through T2 to T3 compared to ‘SJ’. Among these 43 DEGs, 22 displayed significant upregulation, including one IAA gene (Dlo_025130.1), three SRURs and two GH3 genes of the Aux-mediated signaling pathway, 5 GID1 and two DELLA genes involved in the GA-mediated signaling pathway, one AHK gene (Dlo_004406) related to the CK pathway, and one PYL, three BRI1 and one MYC2 (Dlo_003448.1) involved in the ABA, brassinosteroid and JA-mediated signaling pathways, respectively. Twenty-one DEGs were significantly downregulated during T1 to T2 compared to ‘SJ’, including an auxin influx carrier family protein and ARF involved in the Aux-mediated signaling pathway, two-component response regulator ARR-A and ARR-B family proteins related to the CK pathway, and GID1, DELLA, ABF and BRI1 involved in the GA, ABA, and brassinosteroid-mediated signaling pathways. Among the 92 DEGs significantly upregulated or downregulated through T2 to T3 in ‘SX’, 51 were upregulated and 37 downregulated. These genes are related to auxin, GA, CK, ABA, ET and JA-mediated signaling pathways; the SA pathway was not represented in this case.In this study, 237 DEGs related to hormone signaling pathways were identified  and two TCP genes, and seven were only significantly downregulated during T1 to T2, including an APRR family gene, EFL (Dlo_027544.1) and GI (Dlo_024864.1). In contrast, only one gene  showed significant upregulation during T1 to T2 in ‘SX’, whereas AP2 (Dlo_000287.1) and CONSTANS-like 14 (Dlo_031781.1) displayed significant upregulation during T1 to T2. The remaining genes were significantly and differentially expressed during T2 to T3 in ‘SX’, including phytochrome (phy), embryonic flower 1, SOC1–1, CONSTANS-like, TCP, REVEILLE, GID, FKF1 and cryptochrome (cry). Most of these flowering-related DEGs are associated with photoperiod and the circadian clock pathways, such as the COL gene, APRRs, FKF1, phytochrome, and TF TCPs. Moreover, only one gene  involved in the floral meristem pathway was significantly upregulated during T1 to T2 in ‘SX’, yet it showed no apparent change during the flower induction process in ‘SJ’. However, TFL1 and FLC, which controls the PF trait in Arabidopsis and strawberry, did not show differential expression during the floral induction process in our study. These results indicate that photoperiod and circadian clock and floral meristem pathways have significant roles in regulating PF trait formation in longan.A hierarchical heat map was constructed to comparatively analyze the 39 flowering-related genes , and sucrose synthase (SUS) decrease during the floral transition process in SF rose, whereas levels of granule bound starch synthase 1 (GBSS1) and starch synthase 2 (SS2) increase [TPS displays a gradual increase during flower induction [SS2, GBSS, and SUS increase during floral induction in Litchi and Doritaenopsis [Arabidopsis, and loss of TPS1 leads to extremely late flowering in Arabidopsis, even under other inducing environmental conditions [TPS1 was only significantly downregulated in ‘SJ’, indicating that TPS1 may function as an inhibitor of PF traits in longan. Similarly, GBSS1, SS2, and pgm were also only downregulated in ‘SJ’. Conversely, the expression levels of several genes, such as SUS and BAM4, related to sugar biosynthesis were only significantly upregulated in ‘SJ’, indicating that these genes act as activators of PF traits in longan.The transition from dormancy to active bud growth requires sufficient energy . As the bidopsis . Similarbidopsis , lychee bidopsis , and citbidopsis . In contbidopsis . These rincrease . In applnduction . In addiaenopsis , 50. Comaenopsis : TPS1 isnditions . However3), indole acetic acid (IAA) and zeatin ribosides (ZRs) differ during the natural floral differentiation process in ‘SJ’ and that ethephon and GA3 may promote flower formation and fruit setting in ‘SJ’ longan [Arabidopsis, localized accumulation of auxin, which releases auxin response factor5/monopteros (ARF5/MP) from Aux/IAA repression, is the main reason for the emergence of flowers on the flanks of the shoot apical meristem (SAM) [Arabidopsis mutants of ARF2, an auxin-mediated TF, display late flowering [GH3, which maintains auxin homeostasis and promotes the conversion of auxin to amino acids, may increase during floral induction in SF rose and decrease in PF rose [GH3, LAX (auxin influx carrier), IAA (auxin-responsive protein IAA), and SAUR family genes, were significantly differentially expressed during floral induction (T1 to T2 transition) in these two longan cultivars that exhibit differing flowering traits  . Similarem (SAM) . In addilowering . Levels  PF rose . Similarits Fig. . TherefoArabidopsis [Arabidopsis histidine-containing phosphotransfer protein6 (AHP6), a negative regulator of CK signaling, is induced by the MP protein. AHP6 then may diffuse to adjacent sites, where it inhibits meristem initiation, thereby enhancing the auxin phyllotactic pattern [Arabidopsis histidine kinase2 (AHK2), AHK3, and AHK4/CRE1 are three membrane-located receptors that perceive the cytokinin signal [ahk2 and ahk3 variant alleles display an early flowering phenotype [AHK genes displayed significant up- or downregulation during the early floral induction stage in ‘SJ’, respectively, no significant changes were observed in ‘SX’, indicating these genes may participate in regulating floral induction in ‘SJ’.The phytohormone cytokinin (CK) is a regulator of many processes in plants, including cell proliferation and differentiation, shoot and root growth, seed germination, and leaf senescence , 59. Prebidopsis –63. Duri pattern , 65. Aran signal –68, and henotype . In the GID1 and 4 DELLA genes were upregulated in the early floral induction stage in ‘SJ’, and 3 GID1 genes were downregulated. In contrast, 5 GID1 and 2 DELLA genes were upregulated in the early floral induction stage in ‘SX’, and 2 GID1 genes and one DELLA gene were downregulated. These results suggest that the GA signaling pathway may have different functions during floral induction in these two longan cultivars; however, further analyses are required.GA is an important class of plant hormones involved in many aspects of development . In woodMYC2, Pyrabactin resistance 1-like4 (PYL4), and KIN10 (SnRK2.6) also gradually increase in floral buds during flower induction [Arabidopsis, ABA functions as a inhibitor of flowering [PYL12 and SnRK2/2.5 are downregulated, though protein phosphatase 2C25 (PP2C25) is upregulated, during the floral transition [PYL genes were significantly upregulated but PP2C downregulated during the floral transition in ‘SJ’ and ‘SX’, indicating that the ABA signaling pathway may be involved in regulating different flowering traits in these two longan cultivars.Abscisic acid (ABA) also participates in many developmental processes such as seed development, stresses response, and floral transitions , and exonduction , and whelowering . Similaransition . In contArabidopsis thaliana, brassinosteroid-insensitive 1 (bri1) acts as an enhancer of the late flowering autonomous-pathway mutant LUMINIDEPENDENS (LD), and attenuation of BR signaling enhances FLC expression and delays flowering [BSK (Dlo_029469.1), BZR1 (Dlo_010565.1), CYCD3 (Dlo_000396.1 and Dlo_011212.2) and most BRI1-like genes were upregulated in the early floral induction stage in ‘SJ’ , which is widely distributed in plants, plays a role in plant growth and development as well as responses to various stresses –77. In alowering . SimilarK Dlo_02969.1, BZRFLC [Arabidopsis and Triticum aestivum, and mutation of bHLH TF genes represses JA signaling, resulting in late blooms; the JA receptor mutant coi1 also shows early flowering [CTR1 (Atctr1), which act as inhibitors of ethylene signaling, exhibit late flowering, whereas mutations in OsERS2, OsETR2, and OsETR3 cause enhanced ethylene sensitivity and early flowering, indicating that ethylene inhibits flowering in Arabidopsis and rice [CTR1, jasmonic acid-amido synthetase JAR1, MYC2, and bHLH TF genes were upregulated during early floral induction in ‘SJ’, and ERF-1 (ethylene response factor) and TGA1 TF genes were downregulated during early floral induction in both ‘SJ’ and ‘SX’. In summary, hormone biosynthesis and signaling pathways may affect PF trait formation in longan. However, further research is needed on the specific functions of hormone signaling in regulating PF trait formation in longan because of the complex networks of crosstalk between different hormones and reported contradictions between various species.Other hormone pathways such as the ethylene (ET), salicylic acid (SA), and methyl jasmonate (MeJA) signaling also affect floral induction , 81. ForFLC . MeJA aplowering , 84. Mutand rice , 86. In LFY and AP1), and promote flowering [Arabidopsis, strawberry and rose [TFL1 and FLC, which control PF in Arabidopsis and strawberry showed no differential expression during the floral induction process in our study, possibly indicating a different mechanism of PF trait formation between longan and model plants. Thus, it is necessary to investigate the regulatory mechanisms involved in PF trait formation in fruit trees, which is directly linked to production potential. In this study, 39 flower-related genes differentially expressed during floral induction between ‘SJ’ and ‘SX’ were identified , which participates in the circadian clock pathway [Arabidopsis and also exists in other plants such as in Populus deltoides and apple [CO-like genes  were upregulated during the floral induction process in ‘SJ’, whereas GI (Dlo_024864.1) displayed an opposite expression pattern, suggesting that these COL and GI genes may function as activators or suppressors of floral induction in ‘SJ’. Similar to COL genes, two teosinte branched 1/cycloidea/proliferating cell nuclear antigen factor (TCP) genes , which act as CO activators [GAI gene were upregulated during floral induction in ‘SJ’. Pseudo-response regulator (PRR) proteins have the ability to increase CO binding to the FT promoter, leading to FT transcriptional enhancement and early flowering [PRR genes showed changes similar to those of GI, indicating potential functions in PF trait formation in longan. Early flowering (EFL) proteins are a type of circadian clock component with an important role in flowering. For instance, EFL3 inhibits flowering under noninducing photoperiods by blocking the production of GA and expression of FT1 [ELF4 (Unigene4309) increased in ‘SJ’ compared with ‘Lidongben’, indicating that ELF4 may be involved in PF traits and that ELF4 may be a key gene. Similarly, the EFL4-like gene (Dlo_027544.1) functions as a suppressor of floral induction in ‘SJ’. APETALA2 (AP2), a target of miR172 that has been implicated in floral stem cell control [COL14, indicating a promoting role in this process. Guo et al. [LFY, a plant-specific TF, is the central floral meristem identity gene [LFY is an important determinant of flower initiation [LFY acts as a key regulator in the integration of flowering signaling pathways, controls the transition from the inflorescence meristem to the floral meristem, and regulates flowering time [LFY is directly induced by the MP protein, and it then activates expression of the downstream regulator axillary meristems1 (RAX1) and simultaneously inhibits expression of Arabidopsis response regulator7 (ARR7), thereby activating CK signaling and eventually promoting floral meristem formation [CAL), short vegetative phase (SVP), agamous-like 24 (AGL24), and apetala1 (AP1) are induced by LFY to specify floral identify. Indeed, a feed-forward regulatory loop between AP1 and LFY stabilizes floral identity and inhibits TFL1 to promote the transition from the inflorescence meristem to the floral meristem [TFL1 expression is suppressed by AP1 and directly promoted by LFY, indicating that LFY has an inhibitory effect on flower formation [LFY (Dlo_005438.1) was significantly upregulated during T1 to T2 in ‘SX’, though it showed no apparent changes during flower induction in ‘SJ’, suggesting a potential inhibition role in longan PF trait formation.Flowering is a complex process of morphogenesis that is controlled by a complex network involving vernalization, autonomous, photoperiod, GA-dependent, and aging pathways. These pathways, which are both independent and cross-linked, generate positive and negative feedback and combine flowering signals into several key floral integrators , activate floral meristem identify genes  determines floral meristem and floral organ identity in rice [Arabidopsis [In addition to the above genes involved in different pathways, several TFs such as GRAS, MADs, NAC and MYB also play a vital role in regulating downstream floral transition genes –39. Prev in rice . Among t in rice , 107, anbidopsis , 108. MYTFL1 and FLC, which control the PF trait in Arabidopsis and strawberry, showed no significant differential expression during floral induction in our study, possibly indicating a novel mechanism of PF trait formation in ‘SJ’ longan. In addition, the LFY gene may inhibit PF in ‘SJ’ longan, though further analyses are required. This study provides a platform for understanding the molecular mechanisms underlying changes between PF and SF longan genotypes and will aid future studies on PF trait mechanisms of evergreen fruit trees.Research to date on the regulatory mechanisms involved in PF has mainly focused on model plants. As an important trait that is directly linked to production potential, it is necessary to elucidate the regulatory mechanisms involved in PF trait formation in fruit trees. In this study, comparative transcriptome analysis was performed using two longan cultivars that exhibit differing flower phenotypes during the floral induction process. According to our study, the transcriptional differences in ‘SJ’ are mainly concentrated at the early floral induction stage, which is consistent with a previous study showing that red buds (T2 in our study) are the signal for floral induction. Comparing these two longan genotypes, the transcriptional landscape of the T1 to T2 transition differed greatly with regard to key hormones, circadian rhythm, and sugar pathways, as well as TFs. Almost all of the flowering-related DEGs identified are involved in the photoperiod and circadian clock pathways. Unexpectedly, Additional file 1:List of primers used in this study. (XLSX 12 kb)Additional file 2:The sequencing and mapped results obtained from each sample. (XLS 22 kb)Additional file 3:Identification of known genes and new genes in the transcriptome data for eighteen samples. (XLSX 11 kb)Additional file 4:DEGs between ‘SJ’ and ‘SX’ during floral induction. (XLS 11846 kb)Additional file 5:Significantly enriched GO term analysis of DEGs between ‘SJ’ and ‘SX’ during floral induction. (XLSX 22 kb)Additional file 6:Significantly enriched KEGG pathway analysis of DEGs between ‘SJ’ and ‘SX’ during floral induction. (XLSX 15 kb)Additional file 7:DEGs in ‘SJ’ during floral induction. (XLS 7620 kb)Additional file 8:DEGs in ‘SX’ during floral induction. (XLS 7771 kb)Additional file 9:GO terms significantly overrepresented in the eight enriched profiles of gene expression versus the reference set in ‘SJ’ and ‘SX’. (XLSX 71 kb)Additional file 10:KEGG pathways significantly overrepresented in the eight enriched profiles of gene expression versus the reference set in ‘SJ’ and ‘SX’. (XLSX 20 kb)Additional file 11:Sugar-related DEGs in two longan cultivars during the floral induction process. Red indicates the genes that showed upregulated expression; blue indicates the genes that showed downregulated expression. (XLSX 48 kb)Additional file 12:Hormone-related DEGs in two longan cultivars during the floral induction process. Red indicates genes that showed upregulated expression; blue indicates genes that showed downregulated expression. (XLSX 107 kb)Additional file 13:Flower-related DEGs in two longan cultivars during the floral induction process. Red indicates genes that showed upregulated expression; blue indicates genes that showed downregulated expression. (XLSX 26 kb)Additional file 14:GARS, NAC, MADs, MYB TFs in two longan cultivars during the floral induction process. Red indicates genes that showed upregulated expression; blue indicates genes that showed downregulated expression. (XLSX 125 kb)Additional file 15:2 expression ratios; the y-axis indicates the RNA-Seq data ratios. (TIF 262 kb)Linear relationships between qRT-PCR data and RNA-Seq data of related genes. The x-axis indicates the qRT-PCR log


O2max: 71 ± 6 ml ⋅ kg−1 ⋅ min−1) completed three work-dependent TT following ∼30 min administration of 2 mg nicotine gum (GUM), ∼10 h administration of 7 mg ⋅ 24 h−1 nicotine patch (PAT) or color- and flavor-matched placebos (PLA) in a randomized, crossover, and double blind design. Measures of nicotine’s primary metabolite (cotinine), core body temperature, heart rate, blood biochemistry  and Borg’s rating of perceived exertion (RPE) accompanied performance measures of time and power output. Plasma concentrations of cotinine were highest for PAT, followed by GUM, then PLA, respectively (p < 0.01). GUM and PAT resulted in no significant improvement in performance time compared to PLA , with mean power outputs of 264 ± 31, 265 ± 32, and 263 ± 33 W, respectively (p = 0.74). Core body temperature was similar between trials (p = 0.33) whilst HR averaged 170 ± 10, 170 ± 11, and 171 ± 11 beats ⋅ min−1 (p = 0.60) for GUM, PAT, and PLA, respectively. There were no differences between trials for any blood biochemistry  or RPE with mean values of 16.7 ± 0.9, 16.8 ± 0.7, and 16.8 ± 0.8 (p = 0.89) for GUM, PAT, and PLA, respectively. In conclusion: (i) nicotine administration, whether via gum or transdermal patch, did not exert an ergogenic or ergolytic effect on self-paced cycling performance of ∼1 h; (ii) systemic delivery of nicotine was greatest when using a transdermal patch; and (iii) nicotine administration did not alter any of the psycho-physiological measures observed.The use of nicotine amongst professional and elite athletes is high, with anecdotal evidence indicating increased prevalence amongst cycling sports. However, previous investigations into its effects on performance have not used high-validity or -reliability protocols nor trained cyclists. Therefore, the present study determined whether nicotine administration proved ergogenic during a ∼1 h self-paced cycling time-trial (TT). Ten well-trained male cyclists (34 ± 9 years; 71 ± 8 kg;  Presently, the use of nicotine or nicotine-containing substances is not banned by the World Anti-Doping Agency (WADA). Yet, use of nicotine or nicotine-containing substances amongst elite and professional athletes is high and increasing. For example, cross-sectional, self-report data indicate a 25–35% prevalence of smokeless tobacco use, whilst data from anti-doping urine analyses display a detection of nicotine or its metabolites in 23–36% of samples see  to furthAnecdotal reports indicate an increased prevalence of nicotine use in cycling sports. Through its psychostimulatory and sympathomimetic properties, nicotine exerts psychological and physiological effects that should be nootropic and ergogenic . To dateThe 40 km time-trial (TT) in cycling is often viewed as the blue riband event, featuring in national, international , world and Olympic championships. Nicotine is delivered via different routes using a variety of products, which apart from affecting ease-of-use, can result in different nicotine bioavailability and pharmacokinetics . TherefoDeclaration of Helsinki, except for registration in a database, with each participant providing informed, written consent.The study was approved by the Central Regional Health and Disability Ethics Committee (CEN/08/09/056), and conformed to the standards set by the latest revision of the −1) and peak rate of O2 consumption . All participants were non-smokers, and did not habitually use any form of nicotine administration.Ten well-trained male cyclists  volunteered to participate in this study. All participants were competing at a club or national level on a regular basis and maintained a weekly training volume of more than 200 km. According to −1 facing participants.All participants attended the laboratory on five occasions: (1) preliminary submaximal and maximal tests, (2) experimental familiarization, and (3–5) experimental trials. The three experimental trials were completed in a randomized, crossover, double blind design. All visits were separated by 7 days, conducted at the same time of day (±1 h), following >24 h of dietary and exercise control, with participants also having refrained from alcohol and caffeine during this period. All exercise was on an electromagnetically braked cycle ergometer  with participant-specific set up for the seat, handle bars and pedals, which was maintained constant for each trial within a participant. All testing was conducted in a temperate laboratory environment (18–22°C) with a fan-generated airflow of 19 km ⋅ hO2peak. Work rate began at 100 W and consisted of a linear increase at 40 W ⋅ min−1 until volitional fatigue. Expired gases were collected continuously  for the determination of ventilation and O2 uptake (O2). Following this, a linear relationship between the mean rate of O2 during the last 2 min of each submaximal stage and power output was determined and used to calculate a power output which would elicit 80% of O2peak for each participant for the remaining TTs.Following body mass  and height  measurements, participants began a submaximal test that consisted of four consecutive 5-min power outputs: 100, 150, 200, and 250 W, at a self-selected but constant cadence. Following 5 min active recovery and 5 min inactive recovery, a ramp protocol was used to determine The familiarization trial was undertaken to ensure participants were accustomed to the experimental procedures and to minimize learning effects. This trial replicated entirely the experimental trial outlined below.O2peak. Participants were then provided with a standardized snack , and recorded their diet during the 24 h period prior to the first experimental trial. This diet was replicated for each subsequent experimental trial, and in order to further minimize variation in pre-trial metabolic state a standardized meal  was consumed 3 h prior to arriving at the laboratory for the experimental trial, after which no food was consumed. Fluid was encouraged and ad libitum until 3 h prior to the experimental trial.Participants were asked to refrain from exercise between 24 and 48 h prior to each experimental trial. Twenty-four hours prior to each experimental trial, participants attended the laboratory to complete a standardized training ride 60 min in duration at a fixed power output that elicited ∼60% −1, Habitrol, Novartis, New Zealand) or a placebo patch . Participants were also given a factory-calibrated temperature-sensing radio pill  to ingest at this time. For most, this occurred ∼1 h before each participant went to bed. Approximately 10 h later, at 40 min prior to the beginning of the trial, the same independent staff member handed participants a piece of gum to chew for 30 min. The gum was either nicotine gum  or a placebo gum . Participants were asked to chew the gum as directed by the manufacturer; briefly, this involved participants chewing the gum until the flavor became strong (∼1 min), then placing against the cheek until the flavor disappeared (∼2 min). This process was repeated until 30 min had elapsed. Participants were not aware of the research hypotheses, and were informed that the purpose of the study was to investigate the timing of nicotine administration, hence they would be administered three of the following four options: (i) PAT-GUM, PAT-PLA, PLA-GUM, PLA-PLA. Following the third experimental trial, participants were fully de-briefed. The independent staff member was only aware that they were administering intervention A (PAT), B (PLA patch), C (GUM), or D (PLA gum) with results remaining blinded to the authors until data collection was complete, after which disclosure was made.Approximately 10 h prior to each experimental trial, a staff member not involved with the research project placed a patch on the participant between the right shoulder blade and the spine. The patch was either a nicotine patch  before a final blood sample was obtained.Following the pre-trial control described above, participants arrived at the laboratory and were checked that they still had the radio pill in their gastro-intestinal tract. A blood sample was obtained from the antecubital vein (see below), following which participants changed into their cycling shorts and top, shoes and socks. They then received their chewing gum and rested seated for 30 min before another blood sample was obtained. Participants then completed 3 min cycling at each of 100, 150, and 200 W, to allow sufficient warm-up. Immediately on completion of the 200 W bout, the ergometer was set to linear mode based on the formula of gi), Borg’s rating of perceived exertion (RPE) measured using the 15-grade scale, from 6 to 20 , gastro-intestinal body temperature . The tube containing lithium heparin was analyzed within 30 min for determination of pH, bicarbonate and lactate via an automated analyzer .Venous blood samples were obtained from an antecubital vein into two vacutainer tubes , one 4 ml containing lithium heparin and one 4 ml containing clot activator. Following inversion, the tube containing clot activator was allowed to clot at room temperature for 30 min before being centrifuged  at 4°C for 10 min at 805 −1.Due to nicotine’s tendency to fluctuate and relatively short half-life, cotinine, its major metabolite with a longer retention time is preferred, especially for anti-doping purposes . Sample post hoc pairwise analyses were performed using a paired samples t-test (Bonferroni correction where relevant), with statistical significance set at P ≤ 0.05. Partial eta-squared (ηp2) is reported as a measure of effect size, with demarcations of small (<0.09), medium (>0.09 and <0.25), and large (>0.25) effects, respectively (P ≤ 0.05 but ηp2 < 0.09) or II  error. The typical error of measurement as a coefficient of variation (CV) between trials was calculated according to All descriptive and statistical analyses were performed with SPSS software for windows . Descriptive values were obtained and reported as means and standard deviation (SD) unless stated otherwise. Levene’s test was used to ensure data did not differ substantially from a normal distribution. Data were analyzed using two-way (treatment × time) ANOVA for repeated measures. Sphericity was assessed and where the assumption of sphericity could not be assumed, adjustments to the degrees of freedom were made . Where main or interaction effects occurred, ectively . This cop < 0.01, ηp2 = 0.85) and time  but no interaction  were observed, such that the magnitude in concentrations were attained in the following order: PAT > GUM > PLA, and concentrations increased above baseline for GUM whilst concentrations remained constant for PAT and PLA. Participants reported no adverse effects with overnight exposure to nicotine via the transdermal patch or through chewing gum.Plasma cotinine concentrations can be seen in p = 0.71, ηp2 = 0.04) but changed over time  such that power output decreased with time before a characteristic end-spurt; however, this was not dependent on treatment . Time to complete the set work was similar between treatments  with performance times of 63.3 ± 4.1 min, 62.9 ± 4.1 min, and 62.6 ± 4.5 min for PLA, GUM and PAT, respectively. This corresponded to mean power outputs of 263 ± 33, 264 ± 31, and 265 ± 32 W, respectively .Mean power output between treatments and the profile over time can be seen in When viewing the performance trials by completion order, the typical error of measurement as a CV between trials was 2.5 ± 1.2%. By comparison, the change in performance time due to treatments was −0.6 ± 4.4% (GUM) and −1.0 ± 4.8% (PAT).p = 0.60, ηp2 = 0.06) but changed over time  such that heart rate was maintained (∼170 beats ⋅ min−1) until it increased (∼174 beats ⋅ min−1) with the end-spurt; however, this was not dependent on treatment . The core temperature response was similar between treatments  but changed over time  such that core temperature increased until 40% before reaching a relative plateau, with end-exercise values of 38.9 ± 0.7°C; however, this was not dependent on treatment . The RPE was similar between treatments  but changed over time  such that RPE increased progressively from 14.7 ± 1.1 (a.u.) to 18.3 ± 1.2 (a.u.); however, this was not dependent on treatment .The responses for heart rate, core temperature and perceived exertion can be seen in p = 0.99, ηp2 < 0.01) but changed over time  such that pH was decreased following exercise; however, this was not dependent on treatment . The bicarbonate response was similar between treatments  but changed over time  such that bicarbonate was decreased following exercise; however, this was not dependent on treatment . The lactate response was similar between treatments  but changed over time  such that lactate decreased from baseline to pre-exercise and then increased following exercise; however, this was not dependent on treatment .The responses for pH, bicarbonate and lactate can be seen in r = 0.63, p = 0.05) and body mass  for PAT, but not for GUM .In absolute terms, the [cotinine] correlated with performance time  nicotine administration, regardless of delivery method, did not exert any effect  on exercise performance, (2) systemic delivery of nicotine was greater when using a transdermal patch than gum, and (3) nicotine administration did not affect any of the perceptual or physiological measures observed.Several previous studies have observed a performance benefit of 7–17% when nicotine or smokeless tobacco is administered , althoug−1). We  were values below the limit of detection i.e., >0<7.8 ng ⋅ ml−1, the vast majority occurring during GUM. Consequently, it appears as though buccal absorption was not maximized and thus nicotine-rich saliva was swallowed, with subsequent first-pass metabolism. Therefore, we cannot exclude the possibility that the pharmacologic effects varied between PAT and GUM. Another explanation could be an insufficient time for the conversion of nicotine to cotinine, however this appears less likely due to the known rates of absorption and metabolism  metabolite cotinine, was a function of route of administration . Whilst −1). We  and othe−1). We  have replism see .impaired performance. At low(er) doses nicotine proves nootropic/ergogenic whilst at high(er) doses it does not , require consideration whether a fixed or individualized  dose should be administered. Results from the present study support an individualized approach if a systemic concentration determines the resultant pharmacologic effect, as lower [cotinine] was correlated with a higher body mass. Importantly, however, absolute [cotinine] correlated with absolute performance time, indicating that high(er) systemic concentrations of nicotine resulted in an does not , this dodoes not .Nicotine exerts psychostimulatory effects via increased mesolimbic dopamine, and a sympathoadrenal effect through release of the catecholamines . Howevergi of ∼39°C in a temperate environment.Many competitive sporting events, especially endurance-related, take place in warm-to-hot environments/climates. As a systemic venoconstrictor nicotine causes cutaneous vasoconstriction, decreased skin temperature, and systemic venoconstriction . When coFinally, it is worth considering the anti-doping stance. The half-life of nicotine is 1–2 h, which is why cotinine is favored as a biomarker for nicotine intake, particularly as urine samples, as its metabolism is far slower than nicotine  with reduced daily fluctuation . TherefoTM conceived the work, obtained the funding, analyzed and interpreted the data, and wrote the first draft of the manuscript. TM, SH, and SS designed the work. All authors acquired the data and contributed to manuscript revision, read, and approved the submitted version.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.