Disrupting maternal circadian rhythms through exposure to chronic phase shifts of the photoperiod has lifelong consequences for the metabolic homeostasis of the fetus, such that offspring develop increased adiposity, hyperinsulinaemia and poor glucose and insulin tolerance. In an attempt to determine the mechanisms by which these poor metabolic outcomes arise, we investigated the impact of chronic phase shifts (CPS) on maternal and fetal hormonal, metabolic and circadian rhythms. We assessed weight gain and food consumption of dams exposed to either CPS or control lighting conditions throughout gestation. At day 20, dams were assessed for plasma hormone and metabolite concentrations and glucose and insulin tolerance. Additionally, the expression of a range of circadian and metabolic genes was assessed in maternal, placental and fetal tissue. Control and CPS dams consumed the same amount of food, yet CPS dams gained 70% less weight during the first week of gestation. At day 20, CPS dams had reduced retroperitoneal fat pad weight (−15%), and time-of-day dependent decreases in liver weight, whereas fetal and placental weight was not affected. Melatonin secretion was not altered, yet the timing of corticosterone, leptin, glucose, insulin, free fatty acids, triglycerides and cholesterol concentrations were profoundly disrupted. The expression of gluconeogenic and circadian clock genes in maternal and fetal liver became either arrhythmic or were in antiphase to the controls. These results demonstrate that disruptions of the photoperiod can severely disrupt normal circadian profiles of plasma hormones and metabolites, as well as gene expression in maternal and fetal tissues. Disruptions in the timing of food consumption and the downstream metabolic processes required to utilise that food, may lead to reduced efficiency of growth such that maternal weight gain is reduced during early embryonic development. It is these perturbations that may contribute to the programming of poor metabolic homeostasis in the offspring. It has become increasingly clear that early environmental influences can have long lasting impact on the normal development and physiology of the fetus. Factors such as poor maternal nutrition, prenatal stress and exposure to medicinal and social drugs can all have negative health consequences for the offspring that persist into adulthood Due to the wide variations in the types and durations of working schedules conducted in the community, it can be difficult to experimentally expose either human subjects or animal models to ‘shift work’. Nevertheless, shift work is characterised by forced disruptions in the timing of activity, sleep and light exposure, leading to disordered endocrine, metabolic and behavioural rhythms. While it is known from epidemiological and experimental studies that these changes can increase the risk to the individual of developing a myriad of chronic health disorders, it also raises the important question of whether exposure to shift work during pregnancy can also affect the developing fetus.in utero and early postnatal exposure to this protocol negatively affected metabolic homeostasis, with offspring displaying age and gender dependent hyperleptinaemia, hyperinsulinaemia, poor glucose tolerance, insulin resistance and increased adiposity To address this question, we recently exposed pregnant rats to a chronic phase shift (CPS) protocol whereby the photoperiod was reversed twice every week throughout gestation and for 1 week after birth. We demonstrated that In this study we investigated the impact of chronic phase shifts on maternal and fetal hormonal, metabolic and circadian rhythms. Pregnant rat dams were exposed to either a control photoperiod or chronic phase shifts of the photoperiod, and maternal weight gain, food consumption, the circulating hormone and metabolite concentrations, and glucose and insulin tolerance were assessed. We also assessed the impact of this protocol on 24 hour profiles of clock and metabolic gene expression in the maternal and fetal liver.All experiments were approved by the University of Adelaide Animal Ethics Committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Albino wistar female rats aged 7–9 weeks were housed with males (2∶1) until pregnancy was confirmed by vaginal smears for sperm. Upon detection of mating, females were housed individually and maintained on either control lighting conditions  or exposed to repeated phase shifts throughout gestation . The chronic phase shift (CPS) protocol involved manipulating the lighting schedule so that every 3–4 days the photoperiod was reversed . The phoAt day 19 to 20 of gestation, a separate cohort of pregnant dams were killed by decapitation at 2000, 2400, 0400, 0800, 1200 and 1600 h . An additional group of 10 non-pregnant control females of comparable age were killed at 0800 h (n = 10). Major organs including brain, heart, liver, kidney, gastrocnemius muscle, epigonadal fat, retroperitoneal fat, spleen, pancreas, stomach and adrenals were rapidly dissected, weighed and stored in RNAlater®  for gene expression analyses. Trunk blood was collected into lithium-heparin tubes. The number of pups was determined, and the second pup and placenta from each horn were dissected and weighed (L2 and R2). The placentae were separated into the labyrinth and junctional zones, and separately stored in RNAlater®, as was fetal liver. Plasma concentrations of glucose, cholesterol, free fatty acids and triglycerides were assayed by colorimetric enzymatic analysis on a Hitachi automated centrifugal analyzer with the use of kits from Roche Diagnostics . Melatonin, corticosterone, insulin and leptin were assayed by RIA using kits obtained from Buhlmann , MP Biomedicals  and Linco Research  respectively.At day 20 of gestation, separate cohorts of pregnant rats were subjected to intraperitoneal glucose tolerance (IPGTT) and insulin tolerance tests (IPITT). These occurred 26–28 hours after resumption of a normal photoperiod, and 2–3 hours after lights on. For the IPGTT, overnight fasted animals were injected intraperitoneally with glucose . Blood was obtained from the tail vein before and 15, 30, 60, 90 and 120 min after glucose administration. Glucose was analysed by the dehydrogenase method , with additional blood collected into lithium-heparin microvettes at all time points for subsequent insulin assay. For the IPITT, food was withdrawn at the beginning of the test and the rats were injected intraperitoneally with insulin  2–3 hours after lights on. Blood (5 µl) was obtained from the tail vein before and 5, 10, 15, 30, 60, 90 and 120 minutes after insulin administration for glucose determination.free™ kits . First strand cDNA was generated from 2 µg of RNA using Invitrogen Superscipt III reverse transcription kits . Amplification of cDNA was performed on a GeneAmp 7500 Sequence Detection System  in duplicate, using primers designed and optimised in our laboratory  as described previously RNA was isolated from 100 mg of maternal liver, 100 mg of placental labyrinth or 50 mg of fetal liver using TriReagent  according to the manufacturer’s protocol. Only 1 fetus and placenta from each dam was processed for gene expression analysis, corresponding to position L2. Residual contaminating DNA from all samples was removed using Ambion DNAboratory . The exphttp://hutlab.nl), with a probability of P<0.05 considered rhythmic.Statistical analyses were conducted using SPSS v.18 or GraphPad Prism 5. Maternal weight gain and food consumption from the control and CPS groups was fit to a polynomial quadratic equation (Y = B0+B1*X+B2*X∧2). Food consumption data from the LabMaster system was transferred to an Excel document where the amount of food consumed for each rat in each hour throughout gestation was calculated. Litter size was analysed using non-parametric Mann-Whitney tests. Fetal, placental and fetal:placental ratio data was analysed using two-factor ANOVA. Hormonal, metabolite and gene expression data was analysed using two-factor ANOVA (time and treatment as factors) with Bonferroni post hoc analyses to compare individual time points. Area under/above the curve in the glucose and insulin tolerance tests was analysed using t-tests. The probability value used to identify statistical significance was P<0.05. 24 hour data was also fit to a sine curve where the frequency was 24 hours, with the amplitude, peak and trough values determined using Circwave . There was no effect of treatment on the weight of any of the other tissues analysed , with comparisons between groups revealing significant differences in B1 (P<0.001) and B2 P<0.01, , suggestanalysed .There was no effect of treatment on litter size . AnalysiAs expected, plasma melatonin concentrations were elevated at night and low during the day in the control animals, with cosinor analysis revealing a significant fit to the sine curve (P<0.001), and therefore considered to be rhythmic . The propost hoc Bonferroni analyses revealing lower concentrations of this hormone at 2400 h (P<0.001) and elevated concentrations at 0800 h and 1200 h (P<0.001) in the CPS dams.By contrast, plasma corticosterone levels were strikingly different between the two groups. In the control animals, corticosterone secretion was rhythmic P<0.001, , with peAnalysis of the plasma metabolites in the control dams revealed rhythmic concentrations of glucose P<0.05, , insulinpost hoc Bonferroni analyses revealing differences at 1200 h and 1600 h respectively.Despite resumption of a normal photoperiod prior to sample collection, the concentration of these metabolites in the CPS treated dams was profoundly different. Glucose levels changed rhythmically over the 24 hours (P<0.05) with low levels during darkness and subsequently increasing during the following light period. This was reflected in a significant treatment by time interaction (P<0.05) whereby CPS dams had reduced levels at 2400 h (P<0.05), and higher levels at 1600 h (P<0.01). Similarly, insulin concentrations varied differently with time (P<0.01) such that CPS dams had reduced levels at 2000 h and 2400 h (P<0.05). Plasma leptin concentrations were significantly affected by treatment (P<0.001), being reduced during the late darkness period in the CPS dams (P<0.05), whereas free fatty acid concentrations varied differently with time such that CPS dams had reduced concentrations at 1600 h (P<0.001). Both cholesterol and triglyceride concentrations were affected by treatment (P<0.05), with The glucose response to IPGTT did not differ significantly between the control and CPS dams as reflected in the area under the curve  despite changing from a peak at 1934 h to a nadir at 0724 h, in antiphase to that of the controls. The amplitude of this change was greatly reduced in the CPS dams, changing only 4 fold from peak to nadir. While ANOVA revealed no overall effect of treatment (P>0.05), Bmal1 varied differently with time such that CPS dams had increased expression at 2000 h (P<0.01) and decreased expression at 0800 h (P<0.01) and 1200 h (P<0.05).The hepatic expression of P<0.001, . ConversPer1 mRNA in both the control and CPS dam livers was rhythmic (P<0.01), however, the amplitude and time of peak expression varied substantially between the two groups. In the control animals, peak Per1 expression occurred at 2038 h, with an amplitude change of 16 fold. In the CPS dams however, Per1 expression had peak levels at 0922 h, with an amplitude change from peak to trough of only 3 fold. Again there was no overall effect of treatment in the ANOVA (P>0.05), yet Per1 varied differently with time such that CPS dams had reduced expression at 2000 h (P<0.001) and increased expression at 1200 h (P<0.05).The expression of Per2 expression was highly rhythmic and in antiphase to Bmal1 in the control dams changing 19 fold from a trough at 1326 h to a peak at 0126 h (P<0.001). However, in the CPS dams, Per2 expression was not significantly rhythmic (P>0.05), with minimal changes across the 24 hours. There was a significant treatment x time interaction (P<0.001) whereby CPS dams had reduced expression at 2400 h (P<0.001), and elevated levels at 1200 h and 1600 h (P<0.01).Rev-erbα expression in the control dams changed rhythmically over the 24 hour period of sampling, from peak values at 1918 h, decreasing 350 fold to trough values at 0343 h (P<0.001). Alternatively, Rev-erbα expression in the CPS liver dams was not rhythmic (P>0.05), with amplitude changes from peak to trough being only 2 fold. ANOVA revealed a treatment effect (P<0.05), and a treatment x time interaction (P<0.001) whereby CPS dams had reduced expression at 2000 h and 1600 h (P<0.001) and increased expression at 0800 h (P<0.01).6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKfb3) mRNA changed rhythmically over 24 hours, with data from both the control and CPS dams significantly fitting a sine curve  and glycogen phosphorylase mRNA was only rhythmic in the control animals, becoming arrhythmic in the CPS dams. Glucokinase expression was significantly affected by treatment (P<0.05), with post hoc analyses revealing increased expression at 2000 h and 1600 h in the CPS dams (P<0.05 and P<0.001 respectively). There was no overall treatment effect on PEPCK mRNA, however expression varied differently with time (P<0.05) such that CPS dams had decreased expression at 2000 h (P<0.01). Similarly, glycogen phosphorylase was affected by treatment in a time dependent manner (P<0.01), however post hoc analyses with Bonferroni corrections do not reveal a significant difference at any individual time point (P>0.05).P<0.001, . In the Bmal1 and Per1 mRNA was not rhythmically expressed, however in control and CPS animals, the expression of Per2 and Rev-erbα significantly fitted a sine curve  such that decreased expression was observed at 0400 h (P<0.05) and increased expression was observed at 1600 h (P<0.001) in the CPS fetal livers. For Rev-erbα mRNA, there was an overall treatment effect (P<0.05) with post hoc Bonferroni analyses revealing decreased expression at 2400 h (P<0.05) and increased expression at 0800 h and 1200 h (P<0.001) in the CPS fetal livers. Insulin receptor substrate 2 (IRS2) mRNA in the fetal liver was affected by treatment in a time dependent manner . Hsp105 mRNA was affected by treatment (P<0.01), with CPS fetal livers showing reduced expression at 0800 h. Data from both control and CPS animals significantly fitted a sine curve (P<0.05).In the fetal liver,  P<0.05, . Further P<0.05, , however11β-hsd2 and SNAT1 mRNA was examined in placental labyrinth (11β-hsd2 nor SNAT1 expression in the CPS placentas significantly fitted a sine curve (P>0.05), the time dependent changes in the control placentas approached significance (p = 0.06 and 0.09 respectively).The expression of placental abyrinth . There win utero disrupted the timing of circadian and metabolic gene expression in the developing fetal liver.We have previously shown that exposure of pregnant rats to chronic phase shifts of the lighting environment from conception through to early lactation negatively influences a range of metabolic parameters in the subsequent adult offspring ad libitum, the caloric restricted animals caught up so that by the end of gestation there was no significant difference. While this did not affect birth weight, the offspring went on to develop gender dependent hyperphagia, increased adiposity, hyperinsulinaemia and hyperleptinaemia by 5 months of age The changes in weight gain in the CPS dams were unexpected, being reduced during the first week of gestation. However, as gestation progressed, the CPS dams went on to gain weight more rapidly, such that at the end of gestation they weighed only 4% less than the controls. It is conceivable that the reduction in weight gain observed during the early stages of gestation, a critical stage of embryo development, may have mediated the impact of this protocol on the metabolic health of the offspring. Experimental manipulation of weight gain through moderate caloric restriction during the first half of gestation has previously been shown to influence the metabolic health of rat offspring glycogen phosphorylase mRNA in the preceding time points, a key enzyme involved in the release of glucose-1-phosphate from glycogen.At day 20 of gestation, the CPS dams had significantly reduced retroperitoneal fat pad weights and a trend for decreased epigonadal fat weights. Furthermore, maternal liver weight was reduced at the start of the light period, in contrast to the control animals which had elevated liver weights at this time. The reduced fat pad and liver weight was not due to a reduction in these tissues in response to CPS exposure. Instead, the CPS dams failed to increase the size of these tissues in response to pregnancy at the same rate as the controls, as they still had greater fat and liver weights than that of the non-pregnant animals. The reduced liver weights observed at 1200 h may be due to increased hepatic glycogenolysis in response to low plasma glucose, insulin and leptin levels detected through the previous dark period. This is supported by the observation of increased Exposure to the rapid changes in photoperiod has driven changes in the timing of food consumption leading to intermittent grazing rather than consolidated bouts of feeding during the dark period. With each photoperiod shift, the CPS dams are unable to adjust their pattern of food consumption to align with the new phase of darkness, leading to a disrupted profile of food consumption. Repeated exposure to these shifts may have led to decreased food efficiency as reflected in reduced weight gain and adipogenesis, particularly during the early stages of gestation. Weight gain during the later stages of gestation is a reflection of fetal and placental growth, as these tissues account for 25% of maternal weight at term An analysis of the metabolic profile of the CPS and control dams at day 20 of gestation reveal profound changes to glucose, insulin, free fatty acids, triglycerides and cholesterol concentrations in plasma, consistent with disrupted timing of food consumption. In the control animals there were strong rhythms of these metabolites, with the exception of plasma triglycerides. However, in the CPS dams, the concentrations of these metabolites were either in antiphase or arrhythmic. Furthermore, the usual strong relationship between glucose and insulin as observed in the control animals was disrupted. Further analysis of the mRNA of some key hepatic enzymes involved in glycolysis and gluconeogenesis suggest a dampened profile of expression. While none of these enzymes were either up or down regulated, the observed arrhythmicity suggests major changes to these metabolic processes.ob/ob mice, with offspring showing reduced brain weight and altered projections and synaptic inputs to hypothalamic circuits that regulate feeding ob/ob mice, the offspring retain the ability to secrete their own leptin both in utero and postnatally. Nevertheless, the maternal hypoleptinaemia observed in our study may play a role in the programming of perturbed metabolic homeostasis in the offspring.A rhythm in plasma leptin was observed in the control dams, with high levels at night during the time of feeding, and low levels during the day as previously described in non-pregnant rats Rhythmic melatonin secretion is regulated through indirect neural projections from the suprachiasmatic nucleus (SCN) of the hypothalamus to the pineal Nevertheless, we expect that at stages during the protocol, the melatonin secretion would be suppressed, particularly during the periods of 24 hours of light, as light exposure of sufficient intensity completely suppresses melatonin secretion Compared to the aligned profiles of melatonin secretion in the control and CPS dams, there were large differences in corticosterone secretion between the two groups. Like melatonin, corticosterone secretion is regulated through efferent connections from the SCN. Neurons of the SCN terminate on cells of the PVN, leading to rhythmic CRH release, ACTH secretion from the pituitary and downstream rhythmic secretion of corticosterone from the adrenal , which can then affect the transcription of many genes through both glucocorticoid response elements (GRE) or through interaction with other transcription factors Bmal1, Per1, Per2 and Rev-erbα mRNA in the maternal liver was rhythmic, with high amplitude changes in expression from peak to trough. Importantly, the pattern of expression was such that Bmal1 mRNA was in antiphase to Per2, consistent with a functional transcription-translation feedback loop existing in this tissue. When exposed to the CPS protocol however, pregnant dams experienced significant disruption to clock gene expression; the expression of Bmal1, Per2, and Rev-erbα did not significantly fit a sine curve, and the amplitude changes were greatly reduced. Alternatively, within the fetal liver of control animals, Per2 and Rev-erbα mRNA expression was rhythmic with small amplitude changes from peak to trough, but not Bmal1 mRNA expression. Given the importance of Bmal1 in driving rhythmic clock gene expression, and the necessity of this gene in maintaining activity of a transcription-translation feedback loop, it was surprising to then observe robust rhythms of Per2 and Rev-erbα mRNA. Interestingly, CPS fetal livers also rhythmically expressed Rev-erbα and Per2 mRNA, in antiphase to the controls.Given that the CPS protocol manipulates the dominant zeitgeber to the circadian system (ie light), it was of interest to investigate the impact of this protocol on clock genes in both maternal and fetal tissues. Constant light exposure to pregnant dams has previously been shown to dampen the expression of not only melatonin secretion, but also maternal and fetal clock gene expression, which can be regained through nocturnal melatonin administration Rev-erbα transgene, which led to suppression of the core circadian transcriptional feedback loop through suppression of Bmal1 within the liver but not the rest of the body Per2, which maintained an identical profile of expression to that of the controls. This suggests that rather than being solely reliant on a functional transcription-translation feedback loop to maintain rhythmicity, Per2 is instead responding to systemic cues. In the context of the fetal liver developing within a rhythmic maternal system, the ability of Per2 to sense or detect maternal rhythms allows fetal tissue to maintain rhythmicity in the absence of an endogenous circadian transcriptional feedback loop. The molecular mechanisms, however, that transmit this information are as yet unknown, but candidates include core body temperature through heat shock factors, maternal and/or fetal glucocorticoids, maternal melatonin and substrate concentrations. Unfortunately, fetal corticosterone concentrations were not assessed in this study, but may have shed some light on this question, particularly given recent demonstrations of the importance of fetal glucocorticoids in conveying maternal rhythmicity to the fetus Hsp105 also changed rhythmically in the control and CPS fetal livers, but with a different phase of expression. This suggests that heat shock factors may mediate the rhythmicity of Per2 and Rev-erbα in the fetus, particularly given the presence of heat shock response elements in the promoter of at least Per2. It cannot be dismissed that disruptions to the timing of circadian gene expression within the developing fetus, as observed here in the fetal liver, may play some role in the programming of adult metabolic disease.What could be driving the rhythmic transcription of these genes, if not the core circadian feedback loop? Schibler and colleagues developed mice with a conditionally active, hepatocyte-specific We have shown that exposure of pregnant dams to chronic phase shifts of the photoperiod profoundly alters the normal circadian pattern of food consumption, endocrine secretion and metabolite concentrations, as well as metabolic and circadian gene expression in the mother and fetus. Together these disruptions are likely to have a negative effect on the developing fetus. However, the reduced weight gain of the dams during a key stage of early embryo development is likely to play a major role in the previously observed offspring phenotype of increased adiposity, hyperinsulinaemia, hyperleptinaemia and poor glucose tolerance. Intriguingly, the reduced maternal weight gain occurred independently of total food consumption, suggesting that merely changing the timing of feeding and the downstream metabolic processes required to utilise those nutrients, can lead to an altered metabolic phenotype in the adult offspring. In the context of human pregnancies, maternal shift work also severely impacts upon the timing of activity and food consumption. If the effects seen here in a rat model translate into the human situation, then merely disrupting the timing of food consumption may have lasting impacts upon the developing baby, independent of birth weight, which may predispose those offspring to the development of metabolic disorders as adults.Figure S1Schematic of the control and CPS protocols. Upon the presence of sperm in a vaginal smear, female rats were exposed to control lighting conditions , or the CPS protocol whereby the photoperiod was reversed twice every week (right panel). Food and water was provided ad libitum throughout the protocol. Blue line, positive vaginal smear; green lines, time of tissue collections for hormonal, metabolite and gene expression analyses; red block, time of glucose and insulin tolerance tests.(TIF)Click here for additional data file.Table S1Primer sequences.(DOCX)Click here for additional data file.Table S2Cumulative food consumption as measured by the LabMaster system.(DOCX)Click here for additional data file.


Plasmodium falciparum is the species responsible for the most severe form of malaria leading to different complications. Beyond the development of cerebral malaria, impairment of renal function is a mortality indicator in infected patients. Treatment with antimalarial drugs can increase survival, however the long-term effects of malaria on renal disease, even after treatment with antimalarials, are unknown. The aim of this study was to evaluate the effect of antimalarial drug treatment on renal function in a murine model of severe malaria and then evaluate kidney susceptibility to a second renal insult. Initially, mice infected with Plasmodium berghei ANKA achieved 20% parasitemia on day 5 post infection, which was completely abolished after treatment with 25 mg/kg artesunate and 40 mg/kg mefloquine. The treatment also decreased plasma creatinine levels by 43% and partially reversed the reduction in the glomerular filtration rate induced by infection. The urinary protein/creatinine ratio, collagen deposition, and size of the interstitial space decreased by 75%, 40%, and 20%, respectively, with drugs compared with untreated infected animals. In infected-treated mice that underwent a second renal insult, the plasma creatinine level decreased by 60% and the glomerular filtration rate increased compared with infected animals treated only with antimalarials. The number of glomerular cells, collagen deposition and the size of the interstitial space decreased by 20%, 39.4%, and 41.3%, respectively, in the infected group that underwent a second renal insult compared with the infected-treated groups. These functional and structural data show that renal injury observed in a murine model of severe malaria is partially reversed after antimalarial drug treatment, making the kidney less susceptible to a second renal insult.Malaria is a worldwide disease that leads to 1 million deaths per year.  Plasmodium falciparum is the most severe form leading to the development of cerebral damage , respiratory distress, and kidney injury Malaria is a global disease that affects about 500 million people every year; about 1.5 million people die from malaria every year Plasmodium falciparum, indicating that impairment of renal function is a great risk factor for these patients infected with malaria The development of experimental cerebral malaria (ECM) coincides with the accumulation of the parasite in different organs such as the heart, lungs, spleen, liver, gastrointestinal tract, and the kidneys Plasmodium berghei ANKA (PbA), a murine model of severe malaria, is associated with a decrease in cerebral edema after treatment with antimalarial drugs Usually, survival of mice infected with Another important factor is the tubule interstitial injury, which is a crucial step in the progression of renal disease. In this context, tubule interstitial lesions are observed in acute kidney injury induced by malaria infection Two main questions are addressed: (1) Does the treatment of PbA-infected mice with antimalarial drugs lead to complete recovery from AKI even after treatment has been stopped? (2) Are PbA-infected mice treated with antimalarial drugs more susceptible to a second kidney insult? The BSA challenge tubule interstitial injury animal model is used as a second kidney insult. In this animal model, the glomerular flow rate (GFR) is not changed.This work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Ethics Committee of Federal University of Rio de Janeiro (permit number CEUA-CCS-098) and by The Committee on Ethical Use of Laboratory Animals of Fundação Oswaldo Cruz (permit number L004/08).6 erythrocytes infected with PbA in a final volume of 200 µl, as a model of severe malaria; and/or (b) animals were subjected to intraperitoneal injections of saline or 10 g/kg/day bovine serum albumin (BSA) for 7 days, a BSA challenge tubule interstitial injury animal model C57BL/6 male mice (6–8 weeks old) were used. When indicated, the animals were (a) infected or not by intraperitoneal injection of 105d); (2) infected and untreated mice (infected5d); (3) noninfected mice that were treated with antimalarial drugs following the protocol described by Clemmer et al 21d); (4) infected and treated mice (infected+treated21d). Untreated mice and treated mice were euthanized at day 5  and 21, respectively.Two experimental protocols were developed: (1) to determine renal function in PbA-infected mice treated or not with antimalarial drugs ; (2) to 21d); (2) noninfected, untreated, and BSA i.p. injection (control+BSA21d); (3) noninfected, treated, and BSA i.p. injection (control+treated+BSA21d); (4) infected, treated, and no BSA i.p. injection mice (infected+treated21d); (5) infected, treated, and BSA i.p., injection (infected+treated+BSA21d). All animals were euthanized on day 21 p.i.In the second experimental protocol, the animals were randomly divided into five groups: (1) noninfected, untreated, and no BSA i.p. injection  and xylazine (5 mg/kg body weight) before blood collection via cardiac puncture.Parasitemia was determined daily in all infected groups throughout the period in the metabolic cage . For thig for 5 min, and the supernatant was separated and stored at −20°C until use. The levels of protein, urea, and creatinine were determined colorimetrically in urine samples using specific Gold Analisa kits . Blood samples were collected via cardiac puncture in heparinized tubes and centrifuged at 600×g for 5 min to separate plasma. In these samples, the level of plasma creatinine and blood urea nitrogen (BUN) were determined. The glomerular filtration rate (GFR) was determined by creatinine clearance (CCr).After an adaptation period of 24 h, urine volume was determined and collected for the last 24 h before euthanasia on days 5 and 21. Urine samples were clarified at 600×On the days of euthanasia, the kidneys were analyzed histologically as described by Landgraf et al The results are expressed as means ± standard error. Differences between the control and experimental groups were analyzed by two-way analysis of variance followed by the Newman-Keuls test for multiple comparisons.5d group. Urine creatinine was unchanged in all groups tested (data not shown). These effects are correlated to the decrease in CCr, a marker of the GFR (21d group) reversed all these effects. However, the antimalarial treatment did not reverse the effect of infection on the BUN level. In addition, antimalarial treatment (control+treated21d) per se increased the urinary flow, BUN, and CCr, but the serum creatinine level was not changed. The ratio between BUN and serum creatinine was increased in both the control+treated21d and infected+treated21d groups  did not change the UPC ratio, indicating that antimalarial treatment does not promote kidney injury. These results were obtained by analyzing the profile of renal protein excretion using sodium dodecyl sulfate polyacrylamide gel electrophoresis (The urinary protein/creatinine (UPC) ratio is a well-established marker of renal injury 5d group . This inphoresis .5d group) showed a higher number of glomerular cells (21d). Antimalarial treatment (treated21d group) did not change the renal histomorphological parameters measured , the serum creatinine and BUN levels returned to the levels observed in mice subjected only to kidney insult (control+BSA21d and control+treated+BSA21d groups). The serum creatinine level was not changed but BUN was increased in the control+BSA21d group. Moreover, the CCr value was not changed when the animals were subjected to kidney injury (control+BSA21d group) but it was increased when the mice were treated with antimalarial drugs , renal injury was attenuated but still observed. The reason for this relies on exacerbated production of proinflammatory cytokines such as interferon-γ, tumor necrosis factor α, and interleukin-6, -1, and -8 produced during malaria infection. The same cytokines are involved in the pathogenesis of malaria-induced AKI and are correlated with the progression of proteinuria It is well established that malaria-induced renal injury is a consequence of parasite adhesion to renal endothelial cells as well as activation of the host immune response In patients or an animal model with malaria, combined treatment with artesunate and mef loquine is sufficient to abolish parasitemia and attenuate cerebral malaria, contributing to improvement in survival One important question in relation to the treatment is the side effects of antimalarial drugs. Campos et al 21d) and kidney insult (control+BSA21d) groups.An interesting result obtained in the present work is the observation that infected mice treated with antimalarial drugs are less susceptible to a second kidney insult. Antimalarial drugs have been used in the treatment of other pathologies besides malaria. The administration of artemisinin protects against sepsis in mice challenged with lipopolysaccharide by decreasing the secretion of proinflammatory cytokines Malaria and septic shock have important similarities in relation to induction of AKI Plasmodium falciparum remains high, despite the advances in therapy and prevention of disease P. falciparum infection or the combination of different pathologies in endemic areas.The pathology of severe malaria has been extensively explored, however mortality induced by Figure S1Effect of antimalarial treatment on urinary protein. Animals were euthanized on day 5 or day 21 post infection and urine samples were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein analysis was based on the intensity of Coomassie blue staining. Urinary samples were collected from the different experimental groups as depicted in the figure.(TIF)Click here for additional data file.


Atractoscion aequidens, across the Benguela region. The geographical distributions of the two populations, on either side of the perennial upwelling cell, suggest a strong correlation between the oceanographic features of the system and the breakdown of gene flow within this species. Genetic divergence , absence of migrants (less than 1% per generation) between populations and coalescent estimates of time since most recent common ancestor suggest that the establishment of the main oceanographic features of the system (2 million years ago), particularly the strengthening and position of the perennial upwelling cell, is the most likely mechanism behind the observed isolation. Concordance between mitochondrial and nuclear genetic markers indicates that isolation and divergence of the northern and southern Benguela populations of A. aequidens occurred deep in the past and has continued to the present day. These findings suggest that the Benguela Current system may constitute an ancient and impermeable barrier to gene flow for warm-temperate fish species.Contemporary patterns of genetic diversity and population connectivity within species can be influenced by both historical and contemporary barriers to gene flow. In the marine environment, present day oceanographic features such as currents, fronts and upwelling systems can influence dispersal of eggs/larvae and/juveniles/adults, shaping population substructuring. The Benguela Current system in the southeastern Atlantic is one of the oldest upwelling systems in the world, and provides a unique opportunity to investigate the relative influence of contemporary and historical mechanisms shaping the evolutionary history of warm-temperate fish species. Using the genetic variation in the mitochondrial DNA Control Region and eight nuclear microsatellite DNA loci, we identified the presence of two highly divergent populations in a vagile and warm-temperate fish species,  Contemporary patterns of genetic diversity and population connectivity within species are known to be influenced both by historical population processes, and by historical and present barriers to gene flow The Benguela Current, in the southeastern Atlantic, is characterized by cold sea surface temperatures, high productivity levels, and the presence of a perennial upwelling cell that physically divides the system into two contrasting subsystems Atractoscion aequidens (Cuvier 1830) is a migratory, benthopelagic sciaenid fish inhabiting coastal environments (15–200 m depth) along the western coast of Africa, from Mauritania to South Africa, and the eastern coast of Australia A. aequidens an ideal candidate to investigate the influence of oceanographic features on population connectivity. Because demographic changes cannot be inferred from climatic or fossil records, genetic data can provide a valuable source of information in this context. To uncouple the influence of contemporary versus historical processes, we analyzed both mitochondrial DNA (mtDNA) and nuclear microsatellite DNA variation in A. aequidens to investigate: i) present patterns of genetic diversity; and ii) the influence of Pleistocene climatic events on genetic connectivity, phylogeography and demographic history across the Benguela Current system. The data presented here provides the first comprehensive study of the influence of the Benguela Current oceanographic features in the genetic substructuring of coastal marine fish species in the region.Due to the nature of the sampling effort, fin clipping from commercial fishing catches, no ethics approval was considered necessary. Permissions for collecting samples were obtained when necessary .n = 382 – sampling sites LUA, BEN, LUC, NBE, PIN), northern Namibia (n = 7 – HEN) and South Africa   see . All indGenetic variation was screened at one mitochondrial DNA (mtDNA) region and eight nuclear microsatellite loci . The mtDA. aequidensp<0.05 A total of 395 individuals from six different localities  were scrH), private haplotypes (PH), haplotype diversity (h) and nucleotide diversity (π) were calculated for mtDNA CR sequences for each sample location using Arlequin v.3.5.1 φST values between samples were estimated using Arlequin v.3.5.1 A. aequidens.Number of haplotypes (aN), allelic richness (AR), observed and expected heterozygosity (OH and EH), and Wright's inbreeding coefficient (ISF), as implemented in FSTAT v.2.9.3 FST levels of 0.005, 0.02 and 0.05 using two effective population sizes (eN = 500 and eN = 2000), and varying the number of generations (t) accordingly. As the software only supports up to 50 alleles per locus, locus Geelb32 was removed , and all simulations performed for seven loci and six populations (n = 50 to n = 70). Each simulation was run for 1,000 replicates, and power was estimated as the proportion of the 1,000 tests that indicated significant genetic differentiation. After assessing power of the microsatellite dataset, pairwise population genetic differentiation was estimated using Weir & Cockerham's STF estimator STF can be low Dest, which is independent of heterozygosity, in SMOGD v.1.2.5 K, from K = 1–6, using burn-in lengths of 20,000 and run lengths of 80,000 Monte Carlo Markov Chain (MCMC) steps. In each analysis, five independent runs were performed for each value of K to ensure convergence of parameters during the runs. Estimation of the most likely K was performed based on the obtained posterior probabilities of the data From the microsatellite dataset, intraspecific and within-population nuclear genetic diversity was estimated as number of alleles  algorithm implemented in Network v.4.6 DFS0θ = 2N0μ), size after expansion (1θ = 2N1μ) and number of generations elapsed since expansion  were estimated assuming a sudden expansion model, and applying a general CR sequence divergence rate of 3.6% per My for marine teleosts A. aequidens undertakes large-scale migrations eN) using DNA sequence variation, when the nucleotide substitution model and rate are known D and FS tests, in order to date the fluctuation of female effective population size (efN) through time, a strict molecular clock was enforced, using a CR sequence divergence rate of 3.6% per My.Assessment of past population expansion was conducted on the mtDNA dataset using Tajima's m), as well as eN and efN were estimated using the coalescent-based method in Migrate v.3.4.2 m were obtained from M (M = mμ), and estimates of eN and efN were obtained from θ . Inference of immigration rate per generation was obtained by multiplying M and θ (xNem) m were obtained by applying a range of mutation rates: the commonly used 0.1% per generation, but also 0.05% and 0.2% per generation. In addition, in order to estimate recent migration rates (1–2 generations) between the two populations, the coalescent approach implemented in BAYESASS th iteration. Convergence of runs was assessed in Tracer v.1.5 Patterns of average, historical and contemporary connectivity between the northern and southern subsystem populations  from the mtDNA dataset. Two independent runs , using the HKY nucleotide substitution model and establishing the tree prior as the coalescent with constant size, were conducted in BEAST v.1.7.4 A. aequidens individuals (GenBank accession number JX192142-286) were amplified and sequenced for 583 bp of CR. The 144 sequences yielded 51 haplotypes, defined by 60 variable nucleotide sites, 51 of which sites were parsimony informative and 29 representing fixed differences between samples from Angola/Namibia and South Africa (A. aequidens exhibited high levels of haplotype diversity (h = 0.853) and moderate levels of nucleotide diversity (π = 0.005), with consistent levels across samples . Despite a smaller sample size, the southern haplogroup exhibited slightly higher genetic diversity values   .A. aequidens individuals (doi:10.5061/dryad.5gf80), and did not exhibit evidence of amplification errors, conforming to Hardy-Weinberg and linkage equilibrium expectation for the majority of loci and samples  below the level accepted to have no substantial impact on differentiation statistics aN), allelic richness (AR) and expected heterozygosity (EH), were similarly high across all samples  and microsatellite  datasets. For mtDNA, pairwise tests . No significant differentiation was observed among Angolan and Namibian samples  or among South African samples .There was significant genetic differentiation between se tests  indicateFST = 0.055, p<0.05; pairwise FST = 0.006–0.05, all p<0.001), but no significant differentiation within each area  – see Dest , and ranged from Dest = 0  to Dest = 0.268 .A similar pattern of genetic substructuring was observed in the microsatellite dataset, with all samples from Angola being significantly different from all samples from South Africa  and microsatellites , but non-significant variation among populations within groups when the hypothesis of a northern versus southern Benguela subsystem population structure was tested.Hierarchical analyses of the distribution of genetic variance (AMOVA) corroborated the pairwise sample tests, in finding significant variation distributed among groups for both mtDNA  and individuals sampled in the southern Benguela region (ARN and PAL) with no haplotypes shared between regions  to the northern (Angola) population, and 2m = 0.0006 individuals in the opposite direction. Using microsatellite data, both MIGRATE and BAYESASS retrieved very similar results: 1m = 0.0003 and 2m = 0.0002 with a mutation rate of 1×10−4 per generation, and 1m = 0.0019 and 2m = 0.0074, respectively. These findings further support the results from STRUCTURE where no admixed individuals were identified between clusters (eN) and female effective population size (efN) were high and of similar magnitude across the two populations .Estimates of average, long-term and recent migration rates among the two putative populations of clusters . CoalescSimilar likelihoods were obtained for estimates of time since the most recent common ancestor (tmrca) between the two putative populations , using tA. aequidens around southern Africa is composed of two distinct and non-interbreeding populations, the distributions of which appear to coincide with contemporary oceanographic features of the Benguela Current system. There was no evidence of genetic population substructuring within the northern Benguela subsystem (Angola and northern Namibia) population or within the southern Benguela subsystem (South Africa) population, but high genetic divergence was found between the two populations in both mitochondrial and nuclear genomes.Our results present evidence that A. aequidens within each region, so effective long-term passive dispersal by eggs/larvae and/or active dispersal by adults must be occurring. The impact of oceanographic features in population substructuring is dependent on species life history features A. aequidens may contribute to the observed panmixia. Both regional populations have pelagic eggs and an extended larval phase, likely to promote gene flow, but the two populations display divergent adult reproductive migration behavior that appears to be matched to different oceanographic regimes in the two subsystems . Based on adult catch information and the small size at maturity, there is no evidence of a spawning migration in the northern population . With a continuous distribution throughout the northern Benguela boundary region , it appears that this population has evolved an extended spawning season (seven months) to ensure that eggs are widely distributed by the bi-directional currents in the region The northern and southern Benguela boundary regions are characterized by multiple oceanographic features such as fronts , freshwater outflows , and multiple upwelling cells A. aequidens must be linked to a major impermeable barrier to gene flow located between the two putative populations. In the case of southern African A. aequidens, the complete isolation of the two populations, indicated by high levels of population divergence for both mtDNA and nuclear markers, combined with an absence of shared haplotypes or identifiable migrants (migration rates less than 1% per generation), suggest not only that the Benguela Current prevents dispersion in all life stages, but also that this is a long term and on-going effect. The genetic break between northern and southern populations coincides with the main upwelling features associated with the Benguela system, with several upwelling cells along southern Namibian and western South African coasts A. aequidens across the Benguela system.Given the effectively homogenized population structure within each region, the substantial genetic divergence between northern and southern populations of φST = 0.902, microsatellite FST = 0.055: probability of genetic homogeneity for both = p<0.001) were very high for a marine teleost. Marine fish typically have widespread distributions, historically large effective population sizes, high dispersal potential and high fecundity, all of which features promote low average genetic differentiation A. aequidens in the Benguela Current region is linked to the presence of an ancient and impermeable barrier to gene flow.Population genetic divergence values for the pairwise comparisons between samples from the northern and southern populations  around 25 Ka. As estimates of time since expansion based solely on mismatch distribution parameters have severe drawbacks as the only source of information regarding a species' demographic history, as they may be influence by early branching in the gene tree A. aequidens population. Severe declines in many marine fishes are associated with the LGM, with the majority of populations showing recovery after the end of the last glacial period ∼10 Ka A. aequidens does not appear to have been as rapid, but occurred earlier than reported for other fishes. In particular, studies conducted on other species in the southern Benguela region suggest that population growth occurred quite recently, during the Holocene (8-6 Ka) A. aequidens populations than those reported for other species in the Benguela Current region. The timing and rate of A. aequidens population expansion suggests that both populations may have survived the LGM in warm refugia that allowed range and population expansion from early in the interglacial period. Early population expansions pre-dating the end of the LGM have been described in warm-temperate fish species in the northern hemisphere, commonly associated with suitable refugia A. aequidens. Furthermore, a warming period between 40-18 Ky has been reported for the region, which may have contributed to an earlier population expansion of warm-temperate species in the Benguela Current system The mtDNA data  indicated strong support for past population growth, suggesting that both A. aequidens occurred deep in the past and has continued to the present day.The concordance between mitochondrial and nuclear genetic markers showing substantial differentiation, and the lack of indication of any contemporary gene flow, indicates that isolation and divergence of the northern and southern Benguela populations of A. aequidens populations in the Benguela Current region dates from the Pliocene-Pleistocene transition approximately 2 Ma. This transition was characterized by a generalized cooling of world oceans and major changes to circulation patterns, which have been linked to population isolation and vicariant speciation events for many fishes A. aequidens populations.The coalescent simulations indicated that the isolation of


In the training phase, thirty parameters, comprising both cytological descriptors and architectural aspects, were identified. The training set included 6 melanomas with a mean Breslow thickness±S.D. of 0.89±0.48 mm. In the test phase, these parameters were blindly evaluated on a test data set consisting of 25 melanomas, 50 nevi and 50 basal cell carcinomas. Melanomas in the test phase comprised 8 in situ lesions and had a mean thickness±S.D. of 0.77±1.2 mm. Moreover, quantitative FLIM data were calculated for special areas of interest. Melanoma was characterized by the presence of atypical short lifetime cells and architectural disorder, in contrast to nevi presenting typical cells and a regular histoarchitecture. Sensitivity and specificity values for melanoma diagnosis were 100% and 98%, respectively, whereas dermoscopy achieved the same sensitivity, but a lower specificity (82%). Mean fluorescence lifetime values of melanocytic cells did not vary between melanomas and nevi, but significantly differed from those referring to basal cell carcinoma enabling a differential diagnosis based on quantitative data. Data from prospective preoperative trials are needed to confirm if MPT/FLIM could increase diagnostic specificity and thus reduce unnecessary surgical excisions.Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a non-invasive investigation of the skin with subcellular resolution. The aim of this retrospective observational  Melanoma (MM) is a lethal cancer among skin malignancies, with an increasing incidence worldwide Multiphoton excited fluorescence imaging  with near-infrared femtosecond-pulsed lasers enables ex vivo and in vivo non-invasive investigation of the skin with subcellular resolution  The aim of our study was to identify morphologic descriptors for melanoma, referring both to cellular features and to architectural aspects, employing MPT fluorescence intensity and FLIM (MPT/FLIM descriptors), and to establish a specific terminology for their assessment. Subsequently, we evaluated the frequency of these descriptors in 3 different populations comprising melanomas, melanocytic nevi and basal cell carcinomas (BCCs), to establish sensitivity and specificity of MPT/FLIM diagnosis. Finally, the diagnostic accuracy for MM achieved by MPT/FLIM was compared to that of dermoscopy.The study was divided into two parts, a training phase and a test phase. In the former, FLIM images referring to 6 MMs were simultaneously evaluated by 3 observers , for the identification of morphologic descriptors characteristic of MM. Proposed descriptors were discussed and 30 parameters were selected after unanimous approval.In the test phase, the presence/absence of the specified criteria were blindly evaluated on a test data set, by 3 independent observers , who also classified the images into those pertaining to melanoma or "other".Moreover, the mean fluorescence lifetime inside a region of interest corresponding to one representative melanin containing cell of each sample, was calculated on images acquired at an excitation wavelength of 760 nm on 25 MMs, 50 nevi and 50 BCCs samples.The local ethics committee  granted approval of the study protocol before the start of the study. The patients provided their written informed consent.ex vivo samples of 25 MMs, 50 nevi and 50 BCCs were considered. MMs, of the superficial spreading type, included 8 in situ lesions and had a mean Breslow thickness (± S.D.) of 0.77 (±1.2) mm (thickness range 0–4.7 mm). No amelanotic and nodular melanomas or lesions showing histological regression were included, but 3 MMs showed a nodular component. Nevi were removed surgically because of equivocal dermoscopic features. BCCs comprised 31 nodular BCCs and 19 superficial BCCs, and one third were pigmented. In all cases, the clinical diagnosis was confirmed by conventional histopathological examination.The training set included 6 melanomas with a mean Breslow thickness±S.D. of 0.89±0.48 mm. In the test phase In this study we used the MPT DermaInspect® imaging system , which provides intra tissue scanning with subcellular spatial resolution  FLIM was implemented in the DermaInspect® system using a TCSPC module with a temporal instrument response function of approximately 250 ps . Data was collected with time bins separated by 50 ps. A short pass optical filter (Schott BG39) was used to protect the detector  from scattered excitation light. The resulting spectral detection window was ∼ 340 nm –610 nm.The FLIM images were calculated using the software SPCImage (Becker & Hickl GmbH). The raw data was smoothed using a 5 x 5 kernel (corresponding to binning setting 2 in SPCImage) and a single exponential decay was fitted at each pixel. The final image output of the FLIM analysis is a pseudocolor image where the color scale encodes the fluorescence lifetime and image brightness encodes the fluorescence intensity. Images were displayed with a red to blue color scale corresponding to 2 fluorescence lifetime ranges: 0–2000 ps . After finding the correct level, the focus on a specific layer was found by means of the software JLScan_1100. The imaging depth was first pinpointed to the upper layer (stratum corneum). Subsequently, two-dimensional, horizontal images were acquired through optical sectioning parallel to the tissue surface  with an interval of 15 µm. Three-dimensional data sets, called z-stacks, were obtained by sequentially changing the plane of focus (z-level), thus scanning at different tissue depths to a depth of approximately 200 µm.One optical section, consisting of 256×256 pixels, was taken with a scanning time of 25.5 s/frame and an excitation power between 20 and 45 mW. Excitation wavelengths of 760 nm and 820 nm were applied for the assessment of the lesions.When imaging in the dermis, autofluorescence signals from both collagen fibers  and elastin fibers (autofluorescence) can be generated. Separation of these signals was accomplished by an optical filter  that can be inserted into the beam path and which excludes the SHG component of the signal when exciting at up to 820 nm. Imaging was accomplished within 30 minutes from biopsy.FLIM images produced by SPCImage were analysed using a custom-written software package to calculate the mean fluorescence lifetime values for selected areas of interest such as single cells on 25 MMs, 50 nevi and 50 BCCs.Dermoscopic diagnosis was performed on 20-fold and 50-fold magnified images acquired by means of a digital epiluminescence microscope  by 3 observers experienced in dermoscopy according to established dermoscopic criteria for MM The test data set for dermoscopy comprised the same lesions studied by MPT/FLIM . Images were presented in a mixed order to the observers, who evaluated them blinded to the histopathologic diagnosis and without knowledge of any clinical data of patients and lesions. For each lesion, observers were asked to make the diagnosis of MM vs non MM. In the absence of unanimity, the diagnosis was established when 2 out of the 3 observers agreed.t-test. A p-value <0.05 was considered statistically significant. Statistical examinations were performed with SPSS 19.0 for Windows .The frequency of each descriptor in MM, nevi and BCC samples was computed. Sensitivity and specificity values of single descriptors and of the MPT/FLIM diagnosis of MM were evaluated as compared with histopathologic diagnosis. Moreover, odds ratios (OD) were calculated for each descriptor. The agreement between ratings made by 3 observers on the diagnosis and specific patterns of MM (inter-rater reliability) was estimated using Cohen kappa statistics with 95% confidence intervals. Finally, mean values and standard deviations of the fluorescence decay in MM, nevus and BCC cells were calculated and differences were assessed by means of the All image analysis was performed using pseudocolor FLIM images produced by the SPCImage software. For each sample, 15 FLIM images were examined. In total, 375 images of MM, 750 images of nevi and 750 images of BCC were considered.Considering 90 images corresponding to 6 MM stacks, 30 descriptors were identified. Keratinocytes, which are not always recognizable at the surface, are infiltrated by atypical melanocytes. When displaying images using a 0–2000 ps fluorescence lifetime range, MM cells exhibit a short fluorescence decay time ; thus, a carpet of red cells appears at the lesion surface. Based on previous published work To increase contrast for the distinction of image details, a 0–400 ps scale can be applied . Since iDeeper MM layers show ASLCs smaller in size with respect to those observable at the surface . They arThe frequency of MM descriptors in MMs, nevi and BCCs is illustrated in Lower MM layers were characterized by ASLCs, different in size, in 88% of the cases. These frequently showed nuclei of different size, were irregular in shape and irregularly distributed, forming aggregates (72%), sheets (84%) or nests (32%). Invasion of the papillae was visible in half of the cases, whereas nests surrounded by fibers or single ASLCs intermingled with fibers were observable in approximately 10% of the cases. Short lifetime cells, different in size or with an irregular distribution were found in 2 nevi.With regard to structural aspects, an architectural disorder was observed in 24 MMs and in 2 nevi. Non edged papillae were observed in 40% of MMs and in 10% of nevi. These were always associated to edged papillae in nevi, whereas in MMs, edged papillae were recognizable only in 3 cases. The χ square test, calculated on the populations of MMs vs nevi, was significant for all descriptors.The classification of a lesion as MM was made based on the presence of ASLCs in the upper layers and architectural disorder. This approach correctly identified 100% of the cases of MM and produced one false positive in nevi . SensitiThe results of the kappa statistics analysis showed a good reliability of all criteria with kappa values ranging from 0.468 to 1.000.Among the descriptors, the ones with the highest probability to identify a MM (OR  = 1176) were "ASLCs irregular distribution upper layers" and "ASLCs different size lower layers", followed by "ASLCs lower layers" and "architectural disorder" (OR  = 576).Fluorescence lifetime values obtained from a single exponential decay fit to the data (+ s.d.), calculated on 3 representative cells on both upper and lower layers of 25 MMs were 347+159 ps and 338+170 ps, respectively. No difference was observed between these values and those referring to nevi (362+121 ps), whereas for BCC the fluorescence lifetime value was significantly higher (1395+130 ps).”All MMs were correctly diagnosed by dermoscopy (sensitivity = 100%), whereas 9 nevi were diagnosed as MM (specificity = 82%).Instrument-based methods have been introduced as a support to clinical diagnosis for early detection of MM and improvement of its prognosis. Dermoscopy is nowadays widely employed in clinical practice Melanins are the main pigments of both constitutive and light-induced human skin color. They are synthesized in specialized organelles and then transferred to surrounding keratinocytes. Melanin content is generally increased in benign and malignant melanocytic lesions and may represent a clue to their recognition. Melanins differ from other skin fluorophores owing to their short fluorescence decay component. Dimitrow et al. investigated procedures of spectral FLIM on 46 melanocytic lesions, and observed remarkable differences in lifetime behaviour between keratinocytes and melanocytes Distinct morphological differences between MM and melanocytic nevi were detected in a study employing only MPT fluorescence intensity imaging by the same group considering 83 melanocytic skin lesions including 26 MMs In order to assess the potential use of the combination of MPT with an improved FLIM technique in MM diagnosis, we performed an observer-blinded study on melanocytic skin lesions to identify robust MPT/FLIM descriptors enabling diagnostic sensitivity, specificity, and reliability values, high enough to guarantee clinical applicability and provide results better than those obtained by a conventional imaging technique, i.e. dermoscopy.MM cells on the surface of the lesion are recognizable at the first glance: starting the scanning of a malignant melanocytic lesion, these pleomorphic atypical cells entirely occupy the upper sheets and are red colored (short lifetime) when employing a 0–2000 ps color scale. ASLCs appear as single cells, irregularly distributed or aggregated, with a distribution similar to that of ascending melanocytes in histopathological sections.Contrary to what was observed by Dimitrow et al. In benign lesions, melanocytes were typically visualized as small cells, and nevus cell nests were found at the dermoepidermal junction. Dendritic cells, representing a major diagnostic criterion for MM diagnosis according to Dimitrow et al., were observable only in one fifth of MM cases and showed the same morphology of those observed in nevi and BCCs. Short lifetime cells, variable in size, were also found in 6% of BCC cases. However, these cells never showed more than one nucleus, a nucleus with undefined contours or a non-homogeneous speckled cytoplasm, like in MMs; they also did not form aggregates and were regularly distributed.BCCs showed the typical structure characterized by elongated cells with double alignment, nests surrounded by fibers and phantom islands Fluorescence lifetime depends not only on the fluorophore itself, but also on the interaction with the local environment Similarly to previous published studies Most MPT/FLIM descriptors identified in this study showed high sensitivity and specificity values. Moreover, a sensitivity of 100% and a specificity of 98% were reached for the diagnosis of MM. These values were higher than those reported by Dimitrow et al., which were based on a reduced number of parameters, attaining an overall sensitivity of 84% and an overall specificity of 76%. Moreover, a comparison between the diagnostic capability of MPT/FLIM and that of dermoscopy, which at present represents the established diagnostic technique for MM, was performed for the first time in this study.Although the penetration depth of MPT is at present limited to 200 µm, making the evaluation of MM thickness impossible, the MPT/FLIM technique holds great promise for in vivo diagnosis of melanoma, enabling a diagnostic accuracy higher than that achieved by dermoscopy. The nevi imaged in this study had been removed surgically because of equivocal dermoscopic aspects and were difficult to diagnose even by an experienced dermoscopist. Thus, MPT/FLIM used in a preoperative phase would have increased diagnostic specificity and reduced unnecessary surgical excisions.


Perca fluviatilis) and in two of its most common parasites, the acanthocephalan Acanthocephalus lucii and the cestode Proteocephalus percae, collected in the period 2009–2010 from Ružín, a seriously polluted water reservoir in Slovakia. Samples of muscle, liver, kidney, brain, male and female reproductive organs and adipose tissue of fish and both parasites were analyzed for As, Cd, Cr, Cu, Hg, Mn, Ni, Pb and Zn, by ICP-MS. Mean concentrations of individual heavy metals in all fish samples decreased in the order zinc > copper > manganese > mercury > arsenic > chromium > cadmium > nickel > lead. Zinc was found to be the dominant element and its antagonistic interaction with copper was confirmed. The kidney was a key target organ receiving the highest mean concentrations of all analyzed metals, but some metals showed specific affinity for particular tissues. In terms of human health, concentration of Hg in fish muscle, which exceeded more than two-times its maximum level admitted in foodstuffs in European countries, is of great importance and should be taken into account. Bioaccumulation factors (C[parasite]/C[fish tissue]) calculated for all elements indicated much higher detection skills of A. lucii and P. percae parasites than fish organs and hence, present results allow proposing both parasite models as useful tools to monitor aquatic environmental quality. Acanthocephalans, however, seem to be superior for heavy metal monitoring, also demonstrated under experimental conditions. Present results also indicate the decreasing heavy metal burden of the reservoir and its gradual recovery in the course of time.Heavy metal concentrations were determined in 43 perches ( One of the most serious problems is pollution by heavy metals . They ocAccording to the Dangerous Substances Directive of the European Union  dangerouBiomonitoring is a vital and rapidly growing field that uses several biological groups such as phytoplankton, macrophytes, invertebrates and fish, as bioindicators . This ecPerca fluviatilis) and in their most common parasites . An experiment was conducted to compare the metal uptake by these two parasite species. Cadmium was chosen for experimental design as the model metal, because of its highly effective accumulation into the body of parasites. Despite the lowest Cd concentration of all elements assessed in bottom sediments of the water reservoir ([parasite]/C[host tissue]) calculated for this element. We also assessed the possible changes of environmental conditions since mining and metallurgical activities were ceased in the area ten years ago.This work was performed to assess the current state of pollution in the Ružín water reservoir, which has suffered severe contamination by heavy metals by comparing their concentrations in several perch tissues as well as in their parasites. We determined the concentrations of nine heavy metals explaining their tissue-specific distribution in the perch   . It was This territory was historically known for intensive mining and ore processing activities. Bottom sediments of the reservoir have high concentrations of heavy metals  originatCyprinus carpio L., pike-perch Sander lucioperca (L.), European perch Perca fluviatilis L., northern pike Esox lucius L., tench Tinca tinca (L.), Wels catfish Silurus glanis L. and brown trout Salmo trutta fario L. Until recently, perch represented the numerically dominant species of the fish community that could be rather easily caught in the reservoir .The rich ichthyofauna of the reservoir (both in terms of species diversity and population density) is made up mainly by common carp 2.2.P. fluviatilis) were collected nearby the Jaklovce village situated at the mouth of the Hnilec River  and cestodes   specimens. The cestode P. percae was detected in 28 perch indicating a prevalence of 64.7% with a mean intensity of 1.4 ± 1.63 (1–7) specimens in the 43 fish hosts studied. Two perch were free of infection. All specimens were isolated from each fish intestine, washed in ultrapure water, counted and frozen individually for further trace element analysis.Fish were transported in water from the reservoir to the laboratory, where they were killed by severing the spinal cord. The perch were sampled and dissected with the help of stainless steel instruments and MiliQ water. The fork length and wet weight of each fish was recorded prior to dissection. Samples of the muscles, kidney, liver, brain, hard roe  and soft roe (the testis filled with mature sperm) and the adipose tissue were taken from all individuals. These samples were stored individually in glass vials and deep frozen until posterior processing for trace element analysis. Fish digestive tracts were screened for parasites using a stereomicroscope. Acanthocephalans ( percae)  represen2.3.P. percae) and acanthocephalans (A. lucii), respectively per exposure, obtained from perch intestine were several times washed in 0.9% neutral phosphate-buffered saline  and then, in a laminar flow cabinet, immediately replaced into 50 mL sterile culture flasks with graduation  containing 20 mL RPMI-1640 medium  treated with antibiotics  and kept at 16 ± 1 °C in a cool room for 1, 2 and 3 weeks. The cadmium solutions were prepared from a stock solution of 100 mg/L cadmium chloride (CdCl2). Twelve parasite sets  were exposed to two different final concentrations of Cd2+: (i) 6.132 mg/L Cd2+ (high dose) and (ii) 0.6132 mg/L Cd2+ (low dose), while the remaining two sets served as unexposed acanthocephalan and cestode controls cultured only in RPMI-1640 for 3 weeks. The integrity and motility  of worms were monitored under stereomicroscope daily. At the end of each week, two groups of both parasite species  were rinsed with ultrapure water and frozen at −20 °C for subsequent cadmium analysis. The parasites from uncontaminated medium were processed in the same way at the end of the third week. During the experiment all parasites survived, and the integrity of their tissues was preserved.The experiment was designed for a comparison of the metal uptake (cadmium) by two different parasite species. Live adult worms, two and four cestodes  and 1 mL H2O2 (Panreac), at 90 °C in an oven and left overnight, according to the standard protocol used at the “Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB)”. After the complete digestion, samples were diluted with 30 mL of Milli-Q water and then analysed for trace elements in an inductively coupled plasma mass spectrometer (Perkin Elmer Elan 6000). For quality assurance standard reference materials Dogfish  liver (DOLT-3) and muscle (DORM-2)  were used in the analyses. Several analytical blanks were prepared under the same conditions and analyzed along with samples in order to determine the detection limits. Values of all monitored heavy metals are presented in μg·g−1 wet-weight.Fish and parasite samples from the reservoir were analyzed for the presence of nine heavy metals . All materials used in the digestion process were thoroughly acid-rinsed. Samples were weighted (around 100–150 mg wet weight) and digested in Teflon vessels with 2 mL HNO2.5.The data obtained did not meet the requirements for parametric statistical tests. Thus, they were analyzed non-parametrically. Adipose tissue and soft roe were excluded from the statistical analysis due to the insufficient number of samples.The differences in heavy-metal concentrations among the analyzed tissues, between parasite and host tissues, and the two parasite species for each tissue separately were tested. For comparing two independent groups, Mann-Whitney U test was used, for multiple independent groups Kruskal-Wallis ANOVA with multiple comparisons of mean ranks for all groups  was seleet al. [[parasite]/C[host tissue]) and checked for statistical significance with non-parametric Mann Whitney U test or Kruskal-Wallis ANOVA with multiple comparisons of mean ranks for all groups, so-called post hoc multiple comparing /C[muscle]) are of particularly great importance denoting much higher detection ability of parasites than fish muscle, which is the tissue tested more commonly in heavy metal biomonitoring studies. In present study the highest bioconcentration factors were determined for cadmium in A. lucii parasites and muscle and hard roe of perch. Similar results achieved Jankovská et al. [A. lucii in the Jevanský stream near Prague (Czech Republic). A potential of cadmium to accumulate in muscles and gonads determined by bioconcentration factors  was also very high. Slightly different results reported Sures et al. [A. lucii parasites. The highest bioconcentation factors were assessed for copper in muscle (250) and liver (25) of fish. Our data, for a comparison, were lower; BFs for Cu were only 67.7 (muscle) and 3.6 (liver).The bioconcentration factors obtained for almost all metals confirmed the high accumulation capacity of parasites  and Pb (0.07 μg·g−1 and 0.02 μg·g−1), which are the two major pollutants coming from the former mining activities. Lead concentration in the fish liver also decreased (0.08 μg·g−1 and 0.02 μg·g−1) but Cu remained at similar level in both periods. The amount of heavy metals in parasites was also significantly lower in the present study. In acanthocephalans, the highest decrease was detected again for Cu and Pb. Nevertheless, increased mercury concentrations in the fish muscle were found (see above) and, therefore, continuing monitoring is recommended. In addition, high concentrations of arsenic were detected in the muscles and liver of perch, and in the parasites. Since arsenic was produced as a by-product of refining the ores of other metals, such as copper and lead in the erstwhile factories, so it could be assumed that present high concentration of this element estimated in fish tissues and parasites still come from these old industry activities but it is difficult to provide a satisfactory explanation for this arsenic increase.The present study was built upon our earlier work performed at the same Ružín water reservoir by Turčeková et al. . Recent On the whole, comparison of results from both sampling periods seems to point out that heavy metal burden of the Ružín water reservoir appears to be decreasing and a gradual recovery of this ecosystem is taking place in the course of time.


Soil temperature and moisture together explained 48%, 86%, 84%, 54% and 54% of the seasonal variations of soil respiration in the five ecosystems, respectively. There was a significant negative relationship between soil respiration and soil electrical conductivity, but a weak correlation between soil respiration and soil pH or soil organic carbon content. Our results showed that soil CO2 emissions were significantly different among different agricultural and natural ecosystems, although we caution that this was an observational, not manipulative, study. Temperature at the soil surface and electric conductivity were the main driving factors of soil respiration across the five ecosystems. Care should be taken when converting native vegetation into cropland from the point of view of greenhouse gas emissions.The variation of different ecosystems on the terrestrial carbon balance is predicted to be large. We investigated a typical arid region with widespread saline/alkaline soils, and evaluated soil respiration of different agricultural and natural ecosystems. Soil respiration for five ecosystems together with soil temperature, soil moisture, soil pH, soil electric conductivity and soil organic carbon content were investigated in the field. Comparing with the natural ecosystems, the mean seasonal soil respiration rates of the agricultural ecosystems were 96%–386% higher and agricultural ecosystems exhibited lower CO The temporal variations in soil respiration (Rs) can be characterized as diurnal/weekly, seasonal, annual, and centennial in situ in every month of growing season to accurately estimate annual Rs of different ecosystems.The dynamics of the amount of global carbon is the key issue of global warming, and soil carbon pools are correlated with carbon dioxide flux emission from soils Soil temperature, soil moisture and their interaction are regarded as the main controlling factors of Rs 2 production from soil incubation The respiration rates for different ecosystems vary significantly, even between two adjacent plant ecosystems. Raich and Tufekcioglu found that under similar growing conditions, the soil respiration rate was 20% higher for grassland than that for forest Arid regions occupy approximately 20% of the global terrestrial surface, and because of climate change, fire and ecosystem change, arid areas are expanding Tamarix ramosissima (Tr), Haloxylon ammodendron and Reaumuria soongorica (HR) are dominant shrub species in this region Phragmites communis (Pc) is distributed over a large region of shallow underground water. To protect cropland and serve as a reforestation species for alkaline and sandy soils, Elaeagnus angustifolia (Ea) is widely cultivated as a farmland shelterbelt in the XUAR. Winter wheat (Triticum aestivum) (Wh) is one of the main grain crops grown in the XUAR, and is sowed in late September and harvested in August. The phenomenon of CO2 absorption in saline/alkaline soils reported by Xie et al. means that it is important to clarify Rs for different natural and artificial plant communities in the wide expanse of saline/alkaline soils in the XUAR The Xinjiang Uygar Autonomous Region (XUAR) in northwest China covers over one-sixth of China's land area and includes the majority of the country's arid areas The objective of this research was to investigate the effects of ecosystem types from natural communities to shelterbelt and crop ecosystems on Rs, and to clarify the relationships between Rs and soil temperature, moisture, and other environmental factors in a typical arid region. The study specifically addresses the question: What are the differences in diurnal and seasonal variations of Rs among different ecosystems? Particular attention is given to difference between natural ecosystems and agricultural ecosystems.All necessary permits were obtained for the described field studies. The study sites are managed by the Fukang Station of Desert Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. The experiment has received the permits for all the field studies from the Fukang Station of Desert Ecology.The study was conducted in the Sangong River watershed, which is located at the southern edge of the Junggar basin. The study area has an arid climate with mean annual rainfall of 160 mm and mean annual temperature of 6.6°C E. angustifolia shelterbelt (Ea)) and three natural ecosystems (P. communis grassland (Pc), T. ramosissima scrubland (Tr) and H. ammodendron + R. soongorica scrubland (HR))  and nd (HR)) . Three r2 flux system equipped with an LI-COR 8100-103 chamber . For better coverage of the spatial variability of soil respiration in the ecosystem, three plots in each experiment plot (20 m×20 m) were chosen for taking measurements. Taking the average distance between two dominant plants (Da) as a standard, Rs was measured (a) near the plant, (b) at 1/4 Da and (c) at 1/2 Da. Before setting up a PVC collar, litter on the soil surface was cleared and living vegetation inside the collar was clipped from the stem tip. In each ecosystem, nine PVC collars  were inserted into the soil with 3 cm exposed above the soil surface. Rs was measured at two hours interval from 8:00 to 20:00 at clear days every month during the growing season from May to October in 2010. As the winter wheat was harvested in August, the last measurements were made after the harvest at the Wh site. The 0-cm-depth (soil surface) and 10-cm-depth soil temperatures and 5-cm-depth volumetric soil water content (%) were measured automatically with a probe equipped with the LI-8100 system.Soil respiration was measured with an LI-8100 automated soil COSoil samples were collected at 0–30 cm soil depth. Three samples were obtained in each of the three plots, and then mixed up to three replicates according to the plot for the analysis. This work was performed synchronously with the Rs measurement in the five ecosystems. The soil samples were naturally dried and ground to pass through a 2-mm sieve. The pH (1∶5 solid–water ratio) and EC (1∶5 solid–water ratio) were then determined with a Eutech PC700 pH/EC meter , and soil organic carbon (SOC) was measured using the methods described by Bao –1), R is the universal gas constant of 8.31 J mol–1 K–1, and T is the soil temperature (°C). The temperature coefficient Q10 was calculated as described The temperature dependence of Rs was fitted with Arrhenius and exponential functions:An exponential-exponential function was used to describe effects of soil temperature and soil moisture on Rs All statistical linear and nonlinear regression analyses, multiple comparisons including one-way ANOVA and homogeneity of variance tests were performed with SPSS 13.0 software 2 m–2 s–1, respectively. Wh had the highest Rs, while HR had the lowest Rs. When the soil temperature decreased to 5°C at 8:00 in October, Rs for some plots became negative  for Tr, Pc and HR but not for Ea and Wh.The diurnal patterns of soil respiration in the five studied ecosystems were similar, having one-peak curves . The meaRs for Wh, Ea, Tr and HR had a single-peak curve, but that for Pc had a minimum in August owing to the low soil water content (11%) .Rs for Wh, Ea and HR peaked in July and then decreased with temperature. In contrast, Rs for Pc peaked in September and was lowest in October.2 m–2 s–1 for Wh, Ea, Pc, Tr and HR, respectively. One-way ANOVA showed that there were significant differences among Rs values of the five ecosystems (P<0.05)  . Rs for P<0.05) only for the Ea ecosystem when using either of the two models. The regressions explained 49.8% and 49.0% of Rs variation, which were lower than the values obtained using the temperature at 0 cm.For the five ecosystems, the best fitted relationships were obtained using soil temperature at 0 cm depth, which explained 48.3% to 80.9% and 49.0% to 67.8% of the variation in Rs when using exponential and Arrhenius functions, respectively. For the relationship between Rs and temperature at 10 cm depth, the regression was effective  when using the three equations.When soil moisture was taken as a single controlling factor of Rs, linear, quadratic and exponential equations did not explain the Rs variation for Wh, Pc, Tr and HR ecosystems. However, for Ea, the soil moisture explained more than 46% of the Rs variation (R2 = 0.27–0.86) in the five ecosystems (Temperature at 0 cm and soil moisture together could improve the correlation coefficients of the regression equation for Rs (osystems . The resosystems .P<0.05), and the variation in Rs among the five ecosystems could be explained largely by EC (R2 = 0.749). However, the correlation between Rs and SOC content was not significant (P = 0.80).Ea had the highest SOC content and lowest EC . Rs had −2 yr−1 for temperate grasslands, 224 g°C m−2 yr−1 for desert scrub and only 60 g°C m−2 yr−1 for tundra Various ecosystems are known to affect the terrestrial carbon dynamics in different ways 2 m–2 s–1, respectively. These values were close to the soil respiration result of 0.58 µmol CO2 m–2 s–1 obtained for desert ecosystems in northwest China Phragmites communis, the mean Rs was 1.08 µmol CO2 m–2 s–1, which is significantly higher than the rates for Tr and HR in this study. This might relate to the survival strategies of the species. P. communis has phalanx clonal growth morphology and can grow dense roots, rhizomes and aboveground tillers The mean Rs values for the whole growing season in the two natural ecosystems Tr and HR dominated by native species were 0.86 and 0.57 µmol COCultivation activities by humans have greatly affected global carbon sequestration 2 under natural conditions 2 m–2 s–1) was similar to that obtained by Xie et al. at a similar time 2 is absorbed by saline/alkaline soil 2 flux of soil only being observed in natural ecosystems in this experiment might imply that the change from natural ecosystems to agricultural ecosystems will decrease CO2 absorption by soil, and this phenomenon should be noted in addressing global warming and cropland exploration in the XUAR.Xie et al. reported that alkaline/saline soils can absorb COSoil temperature and moisture are usually taken as important factors in controlling soil respiration and they can indeed explain most of the variation in soil respiration R2: 0.48–0.81), the surface temperature might be an appropriate indicator of the fluctuations of soil respiration in the study area. Similarly, Pavelka et al. reported that the soil surface was the most suitable depth for recording the soil temperature because it has the best coefficient of correlation with soil respiration and temperature Temperature can affect almost every aspect of soil respiration 10 is commonly used to describe the sensitivity of soil respiration to temperature among different sites 10 vary widely 10 value of 1.5 10 varied from 1.15 to 1.36 as calculated with the exponential function and from 1.14 to 1.34 as calculated with the Arrhenius function, which suggests that Rs generally increases with temperature; however Q10 has lower sensitivity than the global Q10 mentioned above. Because of the inadequacy of the exponential function in the regression of temperature and soil respiration for Pc, we used the Arrhenius function. Owing to the high level of water–salt stress in a natural habitat, the soil microbial communities, biomass and efficiency were affected by the ecosystem type QP<0.01). Not considering other environmental factors, the soil moisture content correlated negatively with Rs; and when the soil moisture content exceeded 20%, Rs became relatively steady. This phenomenon illustrated that moisture content of 20% might be the threshold for the Ea ecosystem, similar to the findings of Rey et al. Soil moisture is another widely accepted factor affecting Rs at the ecosystem level Because soil moisture only slightly explained variations in Rs, we added temperature and the interactions of temperature with the soil moisture to the Rs–soil moisture exponential-exponential model. It was found that this approach better predicts variations in Rs for all five ecosystems. Similar results have been found in many earlier works As soil respiration is a complex process, many environmental factors including salinity (EC), alkalinity (pH) and nutrient supply could affect Rs. Salinity is a severe problem in arid and semiarid regions, and is usually accompanied with high alkalinity, because of calcium carbonate or hydrolysis of sodium carbonate enrichment in the uppermost soil layers In this study, the EC values significantly decreased for Wh and Ea, mainly because of irrigation management that shifted salt into the drainage channel. There was significant negative correlation between mean Rs in the whole growing season and EC, and it can explain 74.9% of the variation among the five ecosystems for the linear function. This may be attributed to the depressive effects on the microorganisms mentioned above. Similar results for the inhibition effects of salinity on RS have been obtained in many previous works pH in the saline/alkaline soils did not explain Rs variations among ecosystems as well as EC did. Although the regression for the mean Rs in the whole growing season and pH was only significant at the 0.10 level, it may indicate that alkalinity was a depressive factor of Rs. Similar results have been reported that Rs correlates negatively with pH when the pH value exceeds 7.0 In theory, SOC content is another important factor affecting Rs because it can supply substrates to microbial heterotrophs 10) was lower in agricultural ecosystems compared with natural ecosystems. In contrast to the case for many other ecosystems, the SOC content failed to explain variations in Rs among ecosystems. However, EC explained Rs variations among ecosystems, and this has implications for future work, especially studies on the wide spread of saline/alkaline areas in the XUAR. Given the size of the XUAR and other similar arid regions around the world, small changes in soil respiration following conversion of natural ecosystems would have remarkable implications for the regional emissions of CO2 flux to the atmosphere. Proper measurements will be needed to better manage the balance between immediate human needs and maintaining carbon sequestration.Seasonal variations in Rs were well explained by the soil surface temperature but not by the 10-cm depth soil temperature or soil moisture, and the Rs sensitivity to temperature (Q