P<0.05) increased the partial factor productivity of applied nitrogen by 88% and 209% in the early-spring and autumn-winter seasons, respectively, when compared to conventional interval fertigation. Although soils under the treatments optimal interval fertigation and optimal daily fertigation received the same amount of fertilizers, the treatment optimal daily fertigation maintained the relatively stable water, electrical conductivity and mineral nitrogen levels in surface soils, promoted fine root (<1.5 mm diameter) growth of cucumber, and eventually increased cucumber economic yield by 6.2% and 8.3% and partial factor productivity of applied nitrogen by 55% and 75% in the early-spring and autumn-winter seasons, respectively, when compared to the treatment optimal interval fertigation. These results suggested that optimal daily fertigation is a beneficial practice for improving crop yield and the water and fertilizers use efficiency in solar greenhouse.Inappropriate and excessive irrigation and fertilization have led to the predominant decline of crop yields, and water and fertilizer use efficiency in intensive vegetable production systems in China. For many vegetables, fertigation can be applied daily according to the actual water and nutrient requirement of crops. A greenhouse study was therefore conducted to investigate the effect of daily fertigation on migration of water and salt in soil, and root growth and fruit yield of cucumber. The treatments included conventional interval fertigation, optimal interval fertigation and optimal daily fertigation. Generally, although soil under the treatment optimal interval fertigation received much lower fertilizers than soil under conventional interval fertigation, the treatment optimal interval fertigation did not statistically decrease the economic yield and fruit nutrition quality of cucumber when compare to conventional interval fertigation. In addition, the treatment optimal interval fertigation effectively avoided inorganic nitrogen accumulation in soil and significantly ( Protected vegetable production systems have been rapidly developed in recent decades in China. Solar-greenhouse, an unheated plastic greenhouse using solar light energy to make sure that vegetables grow normally, plays more and more important roles in China's vegetable production and supplication during the winter It has become the focus of agricultural field that the efficient water and fertilizer management methods are applied in vegetable production in greenhouse. For example, Cabello et al. However, conventional interval fertigation is still common in intensive vegetable production systems in China. In general, the period between successive irrigation and fertilization events is 7 to 10 days, or even longer. Consequently, the nutrient concentration in the root-zone soil may be in excess of plant requirement for growth on the first day after fertigation, and then decreases gradually to reach deficit levels before the day preceding the next fertigation event, and eventually inhibit crop growth and development Drip fertigation, a technique used to manage soil water and nutrition supplies according to the actual water and nutrient requirement of the plants, can be applied to vegetable production to maintain stable water and nutrition contents in the root zone of crops. It applies frequent and small amounts of soluble fertilizers along with water by exerting the soil buffer characteristic and by reducing the time interval between successive irrigations The efficiency of a fertigation treatment can be evaluated with a combination of the availability and distribution of soil water and nutrients, the growth and development of crop root, and the formation of crop yield. These parameters are relatively simple, rapid, easy to obtain and cost effective. Among these parameters, particular attention should be paid to plant root since the morphology and spatial configuration of root can significantly affect soil water and nutrient transformation, mobilization and use efficiency by plant and crop yield Cucumber is one of the major greenhouse vegetables in China. However, inappropriate irrigation and fertilizing practices have caused soil nutrient imbalance that negatively affects cucumber growth and the reduction of soil water and fertilizer use efficiencies. It is important for farmers to manage soil water and nutrition supplies according to the actual water and nutrient requirement of the plants during greenhouse cucumber production seasons. Thus, the objectives of this study were to investigate how the migration of water and salt in soil, and root growth and fruit yield of cucumber are affected by the daily fertigation based upon the actual water and nutrient requirement of the plants.No specific permissions were required for the described field study.The experiment was carried out in 2011 in a typical solar greenhouse of Fangshan District Agricultural Science Research Institute, Beijing, China , which is an experimental station of China Agricultural University. The field study did not involve endangered or protected species.−1, a field capacity of 20.8% and a bulk density of 1.36 g cm−3, and contained 15.8 g kg−1 organic matter, 0.96 g kg−1 total nitrogen (N), 195 mg kg−1 inorganic N, 193 mg kg−1 available phosphorus (P) and 275 mg kg−1 available potassium (K).The greenhouse was covered with polyethylene film (ground area 56 m×7 m) without supplementary lighting and heating. Daily average soil temperatures and air temperature in solar-greenhouse were shown in −2.The experimental period comprised two growth cycles including the early-spring (ES) (February 1-July 29) and autumn-winter (AW) (August 1-December 25) seasons. The varity of cucumber was Zhongnong No. 26. This varity was a high-quality and largely fruit-bearing cucumber hybrid. The average fruit length and width of this varity were 30 and 3.3 cm, respectively. Due to its high resistance to several diseases  and tolerance to both low temperature and weak light, this varity was very suitable for cultivation in solar-greenhouse. Cucumber seedlings with two leaves were transplanted by hand, before which, soils were incorporated with organic fertilizer  at designed rates , and werThe experiment consisted of three treatments:Conventional interval fertigation (CK): Conventional organic manure  and chemical fertilizer were applied at rates based on average fertilization level used by greenhouse cucumber growers in the suburb of Beijing . A detai−1 in the ES and AW seasons (min) application rates were calculated using the method of soil N balance, where expected yield of solar greenhouse cucumber were 120 and 60 t ha−1 in the ES and AW seasons, respectively. The equation Optimal interval fertigation (OIF): The organic manure  application rate was half the rate conventionally applied per hectare . Based o seasons , respectrecommend is Recommended fertilizer N; Ncrop is crop N uptake; Nsafty is soil Nmin safety margin; Nloss is N loss; Ninitial is soil Nmin in the root zone before transplanting; Nmanure is Nmin from the mineralization of organic fertilizer; Nmineralization is Nmin from the mineralization of organic nitrogen in soil.Chemical fertilizer topdressing/irrigation frequencies and irrigation rates were the same as the CK, except fertilizer topdressing rates. A detailed description of chemical fertilizer topdressing/irrigation rates under the treatment OIF was given in Optimal daily fertigation (ODF): The total amounts of organic manure  and chemical fertilizer were the same as treatment OIF , howeverThe total irrigation amount was the same for three treatments in whole growth period of cucumber. Fertigation was used under transparent thin film for all treatment. Irrigations were applied 16 and 7 times in the ES and AW seasons, respectively. The irrigation rate each time for CK and OIF teatments was 22.5–30 mm according to requirement rule of water for cucumber 2O5:K2O formulations, i.e., 20∶20∶20, 19∶8∶27 and 18∶6∶34, which were used at the early harvest stage , the middle harvest stage  and the late harvest stage , respectively. Fertilizers were applied 12 and 6 times in the ES and AW seasons, respectively. Except different fertigation methods related to different treatments, the same local management practices were applied in all treatments. The experiment was a randomized block design with four replications and the size of each replicate plot was 3.9 m×4.8 m. Each replicate plot had three cultivation furrows and was separated from the adjacent plots by plastic films buried at a depth of 50 cm.The chemical fertilizer used in this study was a special water-soluble fertilizer for cucumber called Shengdanshu . This fertilizer had three N:Pw/v) soil (air-dried) to water ratio using an EC meter and combination glass electrodes . Sub-samples of 12 g fresh soil used to estimate mineral N were submerged into 100 ml 0.01 M CaCl2 solution and shaken for 1 hour to extract inorganic N. The extracts were filtered and analyzed using an continuous flowing analyzer  to determine NO3−-N and NH4+-N contents To evaluate the migration of soil water and salt under different fertigation treatments, soil samples from five cores per subplot were collected five times within a single fertigation cycle during the middle fruit harvest period when daily fruit production was very high in each cropping season. For the ES season, soils were sampled on May 27, May 28, May 31, June 2 and June 4. For the AW season, the corresponding sampling times were October 26, October 27, November 1, November 6 and November 12. Soil samples were taken at 0–15, 15–30 and 30–45 cm depth. Soil samples of each plot at each depth were mixed thoroughly and passed through a 2-mm sieve. Sub-samples of 20 g fresh soil were dried for 12 h at 105°C, and then soil water content was determined at the ratio of water and dry soil weight. Sub-samples of about 300 g fresh soil used to measure EC were air-dried passed through a 1-mm sieve. Soil EC was analyzed from a 1∶5  from the 0–15, 15–30 and 30–45 cm soil layers. For each layer, 9 holes were drilled around the cucumber main root in a shape of 3×3 cross square. The roots in the soil were carefully selected and washed to acquire the roots from different soil layers. Root morphology was analyzed by using fresh roots and a root scanner system . Data were then analyzed with the WinRHIZO root analysis software  to determine the root characteristics, including root length, root surface, root volume and average diameter. The root characteristics could be divided into four classification according to the root diameters aggregated into classes of 0.0–0.5, 0.5–1.0, 1.0–1.5 and ≥1.5 mm. The scanned roots were oven-dried at 65°C until weight constancy and weighed.IWUE, kg mm−1):Y and W represent the economic yield (kg ha−1) and the amount of water (mm) applied to the cucumber during the growing cycle NPFP, kg kg−1):F is the amount of fertilizer N (kg) applied to the cucumber during the growing cycle Economic yield was measured for whole cucumber growth cycles in each plot and translated into economic yield weight per hectare. The ratio of yield to water supply was referred to as irrigation water use efficiency  was done to comprehensively determine the whole effect of different fertigation methods on several root morphologic characters .SPSS 17.0 was used to analyse data. Treatment means were separated using the least significant difference (LSD) test at Soil water content under the treatments OIF and CK increased rapidly and then decreased gradually within an irrigation cycle . HoweverP>0.05) between any two treatments in the 15–30 and 30–45 soil layers in both ES and AW seasons.The EC values in the 0–15 cm soil layer were higher and more variable than those in the 15–30 and 30–45 cm soil layers, suggesting the salts were concentrated in the 0–15 cm soil layer . The CK min) contents in the 0–15 cm soil layer were higher and more variable than those in the 15–30 and 30–45 cm soil layers  to obtain two princioal expressions as follow:1, X2, X3, X4 and X5 represent the root dry weight, length, surface area, average diameter and volume, respectively. The eigenvalue contribution rates of PC1 and PC2 were 65.95% and 31.60%, respectively.Root dry weight, length, surface area, average diameter and volume were used to evaluate the plant root growth. Since there were statistical links among each other of these root properties , we used the principal component analysis (PCA) to transform these correlated root variables  into twoSince the greater values of root dry weight, length, surface area and volume and the thinner root diameter, the root system is more powerful for absorption of water and nutrient, the comprehensive principal component (CPC) was received as follow:1 and CPC values in the ES season, but the highest PC2 values in the ES and AW seasons and PC1 and CPC values in the AW season  between the treatment OIF and CK in the 15–30 and 30–45 soil layers in both ES and AW seasons. However, the treatment ODF significantly decreased the root length in the 15–30 and 30–45 soil layers in both ES and AW seasons, when compared to the CK.In general, root length of four diameter classes  decreased with rooting depth under all fertigation treatments . HoweverN from the treatment ODF were significantly (P<0.05) higher than those from the CK (N from the treatment OIF was significantly (P<0.05) higher than that from the CK in both ES and AW seasons. The treatment ODF increased cucumber economic yield, IWUE and PFPN by 6.2%, 6.1% and 103% in the ES season, and 8.3%, 8.4% and 232% in the AW season when compared to the CK, respectively. The treatment OIF increased PFPN by 88% and 209% in the ES and AW seasons when compared to the CK, respectively.In both ES and AW seasons, cucumber economic yield, IWUE, PFPm the CK . No signP>0.05) difference in soluble sugar in cucumber fruit between the optimal fertigation treatments (i.e. OIF and ODF) and the CK (P<0.05) decreased by the treatment OIF in the ES and AW seasons, and by the treatment ODF in the AW season, when compared to the CK. In addition, vitamin C in cucumber fruit was significantly (P<0.05) increased by the treatment ODF in both ES and AW seasons, when compared to the treatment OIF.In general, there was no significant . The explanation is that although the amount of inorganic fertilizer application was sufficient for plant root growth under the treatment OIF during middle fruit harvest period, it was insufficient for plant root growth during the late fruit harvest period, resulting in significant inhibition of root growth and no significant effect on the cucumber economic yield. For instance, contents of mineral nitrogen in soil laye of 0–30 cm of the treatments CK, OIF and ODF during the late fruit harvest period were respectively 93, 35 and 61 mg kg−1. The contents of mineral nitrogen of the treatment OIF was obviousely less than 50 mg kg−1, which is the lower limit of soil Nmin content for depth 0–30 cm for normal growth and development of cucumber Plant roots are able to make different responses to different water and nutrient contents of soils. Although excessive fertilizer application can increase crop yield, it can inhibit plant root growth due to the high nutrient concentration in root-zone. Furthermore, unused nutrients will accumulate in the soils and finally enhance the potential threat to the environment. In contrast, root growth can be depressed when soil nutrient is in deficit Root growth is closely coordinated with shoot growth Although soils under the treatments OIF and ODF received the same amount of fertilizer , the treRoot function is related to the morphological and physiological characteristics of root. However, this study did not consider the root physiological function, hence, further work is required test the root physiological function to elucidate the mechanism of communication of root and shoot growth.In general, vegetables can provide 80% of nitrate absorbed into the human body On the premise of optimal management of irrigation rate for cucumber, optimal interval fertilization based on nutrient target values, including soil mineral nutrient safety margin and crop uptake, and soil nutrients in root-zone had no significant effect on economic yield, but increased partial factor productivity of applied N. Optimal daily fertigation based on the actual requirement of crops maintained the relatively stable water and nutrients in soils, reduced the spatiotemporal variation of water and nutrients in root-zone and promoted cucumber fine root (<1.5 mm diameter) growth. In addition, optimal daily fertigation maintained significantly higher cucumber economic yield, irrigation water use efficiency and partial factor productivity of applied N than optimal interval fertigation.


Vultur gryphus) influenced the foraging behaviors of a top predator in Patagonia, the puma (Puma concolor), in ways comparable to direct risks of predation for prey to address three questions: 1) Do condors exact a foraging cost on pumas?; 2) If so, do pumas exhibit behaviors indicative of these risks?; and 3) Do pumas display predictable behaviors associated with prey species foraging in risky environments? Using GPS location data, we located 433 kill sites of 9 pumas and quantified their kill rates. Based upon time pumas spent at a carcass, we quantified handling time. Pumas abandoned >10% of edible meat at 133 of 266 large carcasses after a single night, and did so most often in open grasslands where their carcasses were easily detected by condors. Our data suggested that condors exacted foraging costs on pumas by significantly decreasing puma handling times at carcasses, and that pumas increased their kill rates by 50% relative to those reported for North America to compensate for these losses. Finally, we determined that the relative risks of detection and associated harassment by condors, rather than prey densities, explained puma “giving up times” (GUTs) across structurally variable risk classes in the study area, and that, like many prey species, pumas disproportionately hunted in high-risk, high-resource reward areas.Predation risk describes the energetic cost an animal suffers when making a trade off between maximizing energy intake and minimizing threats to its survival. We tested whether Andean condors ( Successful animals forage in ways that minimize intra- and interspecific competition and predation risk landscapes of fearAcinonyx jubatusand red foxes, Vulpes vulpesObserved spatial variation in predation risk has been used to define Kleptoparasitism, the stealing of food from another animal, is a common form of interference competition Puma concolor) is a solitary felid and the top predator throughout Patagonia, a vast 1,000,000 km2, sparsely populated region below latitude 39°S in southern Chile and Argentina (Vultur gryphus) is an IUCN near-threatened species Ovis aries, hooked on fences) or newborn ungulates . Condors are clumsy on the ground and forage only in open habitats The puma (Ursus spp.) only exhibit a seasonal influence on puma foraging , mountain steppe (754 km2), shrubs (138 km2), forests (44 km2), and barren mountaintops (53 km2). Valley steppe was flat steppe habitat 200–600 m asl distributed along the floor of Valle Chacabuco, and characterized by unobstructed views. Mountain steppe described steppe habitats 600–1,200 m asl, and was characterized by steep slopes, ravines, and rock outcrops providing pumas greater cover than valley steppe. Shrub habitats occurred between 200–700 m asl, and were characterized by dense woody understories and canopies up to 8 m tall. Forests habitats were found between 700–1,200 m asl, and were dominated by lenga (Nothofagus pumilio), had relatively open understories, and closed canopies >12 m tall. Barren mountaintops were found above 1,200 m asl, held limited vegetation <6 cm tall, but included rugged topography similar to that described for mountain steppe.Our study was conducted in the southern portion of Chile's Aysén District, immediately north of Lago Cochrane in central Chilean Patagonia , 120 endangered huemul (Hippocamelus bisulcus), 22,000 European hares (Lepus europaeus), and 2,500 domestic sheep 2 in the unfenced portions of valley steppes versus 5,081–5,357 kg/km2 in sheep enclosures in valley steppes; 3,635–9,645 kg/km2 in mountain steppes, 177–716 kg/km2 in shrub habitats, 121–477 kg/km2 in forests, and 0–50 kg/km2 on barren mountain tops. Culpeo foxes  and several scavenger birds, including Andean condors, southern (Polyborus plancus) and Chimango caracaras (Milvago chimango), and black-chested buzzard eagles (Geranoaetus melanoleucus) were common.The study area supported large numbers of puma prey, including approximately 6,500 native guanacos . We defined GPS clusters Odocoileus hemionus), which gain weight at a rate of 0.21 kg/day O. virginianus) Chaetophractus villosus) Chloephaga picta) Prey remains, including hair, skin, rumen, and bone fragments, were used to identify prey species, and the state of prey remains, including the location of bite marks and body parts consumed, were used to determine whether the puma had killed the animal or was scavenging. For ungulates, we defined the kill site  as the location where the rumen was found. The age of guanacos up to 24 months old were determined using tooth eruption sequences in the lower mandible We documented whether each carcass was cached (covered with debris), but did not differentiate between caching efforts . Canopy cover at puma kills was measured with a convex spherical crown densiometer . Each densiometer delineated 24 squares with which to quantify canopy cover, and we subdivided each square into 4 quarters, allowing for a potential of 96 total units. With the densiometer held directly over the kill, the canopy was quantified as % canopy cover, calculated as the variable number of 96 sections in which vegetation was visible.We recorded the presence of condors at puma kills through direct sightings, using remote cameras at kills, and/or through associated signs, including feathers, droppings and footprints. We did not attempt to determine whether condors were present at kills investigated more than two weeks after the puma left the area, when signs were more difficult to interpret.We defined the handling time as the total hours from the first to the last GPS location recorded by the puma collar within 150 m of the kill site, even when the puma moved away from the kill site and then returned during the duration of actively feeding at the site . We only quantified handling time for the subset of kills in which Argos and GPS performance allowed us to accurately identify the start and end of each kill, which required continuous GPS locations leading up to and away from a kill.We defined the search time as the total hours between the last GPS location within 150 m of a kill site and the first GPS location within 150 m of the subsequent kill site. Thus we divided puma activity into 2 phases: searching and handling, choosing not to differentiate from among the other behaviors that occurred within each phase . For kill sequences in which Argos and GPS performance allowed us to accurately identify the last point of the first kill and the first point at the next kill, we calculated the distance pumas moved between sequential kill sites in two ways. First, we summed the straight-line distances between sequential locations gathered by the GPS collars for the duration of the search time. Second, we corrected the summed straight-line distances by applying a correction factor of 1.64, calculated by walking a subset of random 2-hr segments of puma trails to better estimate the distances pumas travel in the field We calculated kill rates using two different methods. First, we quantified total kill rates (kg/day) for all prey killed for pumas monitored continuously for a minimum of four weeks. Second, we calculated ungulate kill rates  for the same time periods, for comparison with kill rates for pumas in North America published in Knopff et al. We used a multivariate approach to test whether prey weight (in kg), the presence of condors, canopy cover, and whether the kill was cached carried predictive power in determining handling time. We used pairwise coefficient correlations, and a correlation cut off of 0.5, to test for significant correlation between independent variables. The presence of condors and canopy cover proved significantly correlated, and we selected the presence of condors to be included in the final analysis because it yielded the highest correlation with handling time. In the end, three variables remained to run the model, and we included individual pumas as a random effect to account for variable samples across pumas. Because the data were discrete, we employed a generalized linear model (GLIM) and a Poisson distribution (logarithmic link function) to account for the fact that time at a carcass could not be negative.We then used a GLIM to test whether handling time or prey weight explained variation in the search time immediately following that kill . We incorporated puma as a random effect to account for variation in prey availability among pumas and for variable number of samples from different animals. We repeated the analysis twice, first with all prey data, and second with only the ungulate prey data to re-assess the relationship in case small prey might have biased results.For every carcass that a puma abandoned >10% of the edible meat, we defined the GUT as the handling time . We used a >10% threshold because it increased the sensitivity of our method by allowing us to include smaller ungulate prey that could be eaten more quickly. We expected that any effects on GUTs that we detected at kills with >10% meat remaining would be magnified should we limit our analysis to kills at which pumas abandoned a larger amount of meat. Based on our definition, the smallest GUT was 2, where a puma remained for 2 hours, and the largest GUT was 232, which was the longest period a puma was associated with a carcass. While our measurement may at first seem counterintuitive because traditionally, the larger the GUD, the greater the risk, in our research, the smaller the GUT (the shorter the time at the carcass), the greater the implied risk.sensu predation risk in hWe defined risk classes categorically, by combining two risk scores. The first score reflected the relative risk of harassment by condors we assigned the 5 habitat types found in the study area, and the second score reflected apparent risk as quantified by the spatial distribution of condor activity on the landscape. We first calculated a correlation coefficient of these two variables and determined they were independent (coefficient  = 0.04). Habitats were assigned risk factors  based upon carcass visibility for condors (habitat descriptions are found in the study area description). We assigned forests a risk of 1, shrubs 2, open mountain steppe and Barren mountaintops 3, and open valley steppe 4. We then plotted the locations of kills detected by condors and used a fixed kernel density estimator We calculated prey densities for each habitat type using direct counts for huemul and sheep, and Distance sampling We monitored 8 pumas using Argos-GPS technology , and 1 puma using the stored GPS data in the collar because its Argos capabilities failed (Telonics collar), for a mean of 9.33±5.66 months. We conducted field investigations of Argos-relayed GPS clusters within 11±12  days of the time the puma left the area. We did not use the stored data in the malfunctioning collar in this calculation; site investigations for this animal were conducted on average 792±87  days after the puma had left the area Pumas abandoned an estimated 29±27 kg of meat at each ungulate kill (range 0 – 75 kg). We documented condors at 43% of 296 ungulate kills in which their presence could be determined with certainty. For kills in which handling time could be quantified, pumas abandoned 133 of 266 ungulate kills (or 50%) where >10% of edible meat remained after 1 night, and an additional 56 (or 21%) after 2 nights.n = 340), and 69.5±51.4 hrs for only ungulate kills (n = 229). Neither sex  nor the presence or absence of kittens with females  were significant predictors of search time. The mean distance traveled between sequential kill sites was 19.83±19.23 km (32.52±31.55 km when a correction factor was applied). Males and females moved similar distances , as did females with and without kittens .Mean search time for all prey was 70.2±51.4 hrs  moved into Argentina before 4 weeks of continuous monitoring 8.7±7.5% . The remF1,265  = 18.11, P<0.0001), the presence of condors , and whether the kill was cached  all proved significant influences on handling time. The model explained 63% of the variation in handling time, of which prey weight explained 18%, the presence of condors, 14%, and whether the kill was cached, 12%. Handling time increased with prey weight and caching behaviors, but decreased with the presence of condors.Prey weight  and the analysis using only ungulate prey . Prey weight did not affect search time for the ungulate data  or prey species combined .Shorter handling times correlated with shorter search times in both the analysis using all prey , and were significantly smaller in the areas of highest risk of harassment by condors  whereas relative prey density was not . Puma kills were distributed disproportionately among the riskiest areas more than expected given their distribution in the study area  (GUTs differed among risk classes ( condors . Using 2<0.0001) . Kills wOur research provides evidence that scavengers exact foraging costs upon top predators, and that top predators are aware of and responsive to the costs associated with detection and harassment by competitive scavengers. The fact that pumas abandoned >10% of the edible meat at 50% of large carcasses they killed after a single night of feeding, and never returned to the carcass to see whether meat remained to be eaten, was strong evidence that pumas were aware of the inherent risks of losing carcasses to condors in open habitats. The additional abandonment of 21% of large carcasses with >10% edible meat remaining after only two visits further supported this argument.Puma kill rates for ungulates only (in kg/day) in our study area (12.49 kg/day) proved 50% greater than those reported by Knopff et al. Canis lupus) by shifting their activities into more structured environments and avoiding open habitats where they are most at risk of losing a kill Pumas in North America minimize losses to bears and gray wolves (sensu whiteback griffon vulture, Gyps africanus,Individual condors can ingest approximately 1.5 kg of meat per feeding bout (The question remains, however, as to why pumas abandoned their kills in the first place. We have discussed the avoidance of condor harassment as the potential mechanism driving this behavior–perhaps mobbing condors are difficult to defend against for a solitary predator. Alternatively, condors might not be driving the behavior, but taking advantage of abandoned carcasses. Historic puma persecution in Patagonia was so high as to result in puma extirpation through most steppe grasslands landscapes of fearIt is particularly important that puma GUTs were better explained by relative risk of harassment by condors than by relative prey density. This is strong evidence that top predators suffering energetic costs associated with kleptoparasites exhibit behaviors similar to prey species foraging in We believe our work also lends important insights for understanding predator-prey theory and population dynamics of endangered prey species affected by predation. Our results support previous suggestions that simplistic single prey-single predator models are unable to accurately predict the dynamic consequences of predator-prey interactions in natural systems e.g., . In our


The results of prostate specific antigen (PSA) and digital rectal examination (DRE) screenings lead to both under and over treatment of prostate cancer (PCa). As such, there is an urgent need for the identification and evaluation of new markers for early diagnosis and disease prognosis. Studies have shown a link between PCa, lipids and lipid metabolism. Therefore, the aim of this study was to examine the concentrations and distribution of serum lipids in patients with PCa as compared with serum from controls.Using Electrospray ionization mass spectrometry (ESI-MS/MS) lipid profiling, we analyzed serum phospholipids from age-matched subjects who were either newly diagnosed with PCa or healthy .We found that cholester (CE), dihydrosphingomyelin (DSM), phosphatidylcholine (PC), egg phosphatidylcholine (ePC) and egg phoshphatidylethanolamine (ePE) are the 5 major lipid groups that varied between normal and cancer serums. ePC 38:5, PC 40:3, and PC 42:4 represent the lipids species most prevalent in PCa as compared with normal serum. Further analysis revealed that serum ePC 38:5 ≥0.015 nmoles, PC 40.3 ≤0.001 nmoles and PC 42:4 ≤0.0001 nmoles correlated with the absence of PCa at 94% prediction. Conversely, serum ePC 38:5 ≤0.015 nmoles, PC 40:3 ≥0.001 nmoles, and PC 42:4 ≥0.0001 nmoles correlated with the presence of PCa.In summary, we have demonstrated that ePC 38:5, PC 40:3, and PC 42:4 may serve as early predictive serum markers for the presence of PCa. Prostate cancer (PCa) is the most commonly diagnosed cancer in men and the second leading cause of cancer deaths in men in the western world Screening for prostate cancer is controversial in light of the fact that the two major screening methods for PCa, the digital rectal examination (DRE) and the serum prostate-specific antigen (PSA) test, have limitations Lipids play an important role in biological functions, including membrane composition and regulation, energy metabolism, and signal transduction http://www.promeddx.com). Coded specimens were sent in a frozen state, and the laboratory personnel were blinded as to which of the specimens was from patients or normal individuals until after all of the clinical data and laboratory results became available. Initially, we analyzed the lipid profiles of 154 total serum samples: 77 from prostate cancer patients and 77 from normal subjects. For further statistical analysis, we divided serum samples into two groups: Samples from individuals 50–60 years in age and 61–70 years in age. As we were conducting an age-matched study, we excluded samples from those outside of the two age groups, which resulted in 76 normal (one sample data had an error) and 57 PCa samples. The study has been approved by the institutional review board. For detail medical history of PCa patient please refer to This study was approved (expedited) by Memorial University Medical Center (MUMC) human subjects and ethics committee. ProMedDX, Massachusetts provided all serum samples ; the method is an adaptation of the method described by Bligh and Dyer Data was processed using mass-spectrometer-specific software in conjunction with Excel.An automated electrospray ionization-tandem mass spectrometry approach was used, and data acquisition and analysis were carried out as described previously Unfractionated lipid extracts were introduced by continuous infusion into the ESI source on a triple quadrupole MS . Samples were introduced using an autosampler  fitted with the required injection loop for the acquisition time and presented to the ESI needle at 30 µl/min.+ ions in positive ion mode with Precursor of 184.1 (Pre 184.1); PE and lysoPE, [M+H]+ ions in positive ion mode with Neutral Loss of 141.0 (NL 141.0); PI, [M+NH4]+ in positive ion mode with NL 277.0; PS, [M+NH4]+ in positive ion mode with NL 185.0; PA, [M+NH4]+ in positive ion mode with NL 115.0; CE, [M+NH4]+ in positive ion mode with Pre 369.3. SM was determined from the same mass spectrum as PC (Pre 184.1 in positive mode) Sequential precursor and neutral loss scans of the extracts produce a series of spectra with each spectrum revealing a set of lipid species containing a common head group fragment. Lipid species were detected with the following scans: PC, SM, and lysoPC, [M+H]th set of mass spectra were acquired on the internal standard mixture only. Peaks corresponding to the target lipids in these spectra were identified and molar amounts calculated in comparison to the internal standards on the same lipid class. To correct for chemical or instrumental noise in the samples, the molar amount of each lipid metabolite detected in the “internal standards only” spectra was subtracted from the molar amount of each metabolite calculated in each set of sample spectra. The data from each “internal standards only” set of spectra was used to correct the data from the following 10 samples. Finally, the data were corrected for the fraction of the sample analyzed and normalized to the sample “dry weights” to produce data in the units nmol/mg. The result of this analysis provided a total of 354 potential lipids for early identification of the presence of PCa.The background of each spectrum was subtracted, the data were smoothed, and peak areas integrated using a custom script and Applied Biosystems Analyst software, and the data were corrected for overlap of isotopic variants (A+2 peaks). The lipids in each class were quantified in comparison to the two internal standards of that class. The first and typically every 11To identify potential models using the 354 lipids that were identified, the analysis involved multiple iterations of “best subsets” logistic regression. The analysis was performed as frequently found in “high through-put” data analysis, as limiting models to no more than 3 lipids is equivalent to a genomics problem of over seven million potential biomarkers. Examples of this type of analysis are well-documented Please see flow To identify specific serum lipids species associated with PCa, we performed MS analyses. Given the necessity of simultaneously comparing hundreds of lipids, we incorporated the false discovery rate (FDR) into our analyses We next determined that odds ratio and relative risk for the 31 lipid species identified by MS. We next evaluated whether ePC 38:5, PC 40:3, and PC42:4 could be used as a diagnostic test for PCa based on a sensitivity analysis . Using lCurrently, the major problem in PSA testing is either over- and/or under- diagnosis. On one hand, nearly 15–25% of men have PCa even though their PSA levels are normal (4.0 ng/mL or less) The report on the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening trial notes that screening was not associated with a reduction in PCa mortality during the first 7 years of the trial . These results support the validity of the recent U.S. Preventive Services Task Force recommendations against screening all men over the age of 75 years Studies suggest that certain genetic events that can lead to malignant progression may only occur in cancer precursors (“genetic events indicative of precursor PIN”), and not in non-precursor prostatic intraepithelial neoplasia (PINs). Our previous study Multiple studies have shown an association between PCa risk and diet. For example, Norrish and colleagues demonstrated that dietary fish oils may lower PCa risk, possibly through inhibition of Arachidonic acid-derived eicosanoid biosynthesis For detecting several of the fatty acids, measuring the fatty acid composition of serum phospholipids may give a better reflection of actual consumption of dietary fat than dietary assessment techniques. In fact, fatty acids in serum reflect dietary fat intake in the post-absorptive phase, so processes that affect the bioavailability of fatty acids, such as their transport, excretion, and metabolism, are taken into account In conclusion, because of consistency and robustness, specific phospholipids identified in our study fit the criteria for a phase 1/2 markers Data S1(XLSX)Click here for additional data file.Data S2(XLSX)Click here for additional data file.


Prunus and Fragaria permits detailed comparisons between these genera and the recently released Malus × domestica genome sequence.Comparative genome mapping studies in Rosaceae have been conducted until now by aligning genetic maps within the same genus, or closely related genera and using a limited number of common markers. The growing body of genomics resources and sequence data for both Prunus and/or Fragaria reference maps, and physically to the Malus genome sequence. Markers in common for Malus and Prunus, and Malus and Fragaria, respectively were 784 and 148. The correspondence between marker positions was high and conserved syntenic blocks were identified among the three genera in the Rosaceae. We reconstructed a proposed ancestral genome for the Rosaceae.We generated a comparative analysis using 806 molecular markers that are anchored genetically to the Malus and Prunus was much higher than any reported genome comparisons in plants, suggesting that small inversions have played an important role in the evolution of these two genera or of the Rosaceae.A genome containing nine chromosomes is the most likely candidate for the ancestral Rosaceae progenitor. The number of chromosomal translocations observed between the three genera investigated was low. However, the number of inversions identified among  It has been postulated that the poor phylogenetic resolution along the backbone of the Rosaceae phylogenetic tree suggests a rapid evolutionary radiation of lineages within the family, corresponding to a relatively recent divergence of the genera [Economically, the Rosaceae is one of the most important plant families  comprisio x = 17 . Four suocarpus) . A recenocarpus) . Prunus e genera . Possible genera ,4,5. A bet al. [Solanum tuberosum) and tomato (S. lycopersicum syn. Lycopersicon esculentum) and found remarkable conservation of genome structure, with only a few regions where paracentric chromosomal rearrangements could be identified. More recently, Wu and Tanksley [Comparative mapping has been carried out in a number of economically important plant families including the Poaceae, Solanaceae, Brassicaceae and Fabaceae -10. In tet al.  studied Tanksley  reportedArabidopsis thaliana and Capsella rubella has been observed, with gene repertoire, order and orientation highly conserved [Arabidopsis chromosome I, with just 3 rearrangements identified between the chromosomes of the two species [Arabidopsis and Brassica oleracea, rates of chromosomal rearrangements were shown to be much higher [Prunus and Arabidopsis [In the Brassicaceae, almost complete genome colinearity between onserved  and like species . Howeverh higher . Betweenbidopsis -19, but Prunus species are essentially collinear, for example in peach and apricot [Malus, Pyrus and Eriobotrya were shown to be highly collinear [et al. [Malus and Prunus and found strong evidence that single linkage groups in the diploid Prunus were homologous to two distinct homeologous linkage groups in the amphitetraploid genome of Malus. Vilanova et al. [Prunus  and Fragaria  and they identified numerous chromosomal translocations and rearrangements that occurred in the 29 million years since the genera diverged from a common ancestor. They also found clear cases of conservation of chromosomal synteny, and reconstructed a hypothetical ancestral Rosaceae genome composed of nine chromosomes.Comparative genome studies in the Rosaceae have so far been based on the alignment of genetic maps within the same genus, or amongst closely related genera, using small sets of orthologous markers. These studies showed that the genomes of  apricot ,21, and  apricot ,23. Simiollinear -26. Only [et al.  compareda et al.  comparedMalus × domestica was recently sequenced using a whole genome shotgun approach [M. × domestica genome and those of other genera of the Rosaceae, there are a large number of genetic markers available for Rosaceous species. Recently, Cabrera et al. [Prunus reference map. Because of the conserved nucleotide sequence and low or single copy presence across species, the COS sequences are particularly useful for comparative genome studies between related species [Prunus, members of two different plant families in the eurosid clade [Fragaria reference population.Whole genome sequencing using next generation technologies has now become accessible to the broad scientific community. In the Rosaceae, the genome of approach . The anaapproach . Althouga et al.  reported species . The Rosid clade . Also a Prunus and Fragaria [Fragaria, Malus and Prunus. The locations of molecular markers on the Prunus and Fragaria genomes were determined through bin mapping [Malus × domestica 'Golden Delicious' genome [Malus genome. The findings allowed us to hypothesize about genome evolution within Rosaceae, and to reconstruct its ancestral genome for the family.In this paper, we compared the apple genome sequence with conserved molecular markers previously mapped in Fragaria ,30,31, a mapping ,33, and ' genome . The comPrunus-Malus sequences by aligning all available Prunus EST sequences  amplified a single band, five (3.2%) amplified a length polymorphism between the parents of the bin set that was scored by agarose gel electrophoresis, 20 (12.9%) gave no amplification product, and the remaining four (2.4%) produced multiple fragments. Of the 126 single band markers, those that produced amplicons between 100 and 300 bp (107) were analysed by high resolution melting (HRM). In total, 60 exhibited a clear segregation pattern, enabling their localization on T×E bin map, 17 were monomorphic, and 30 revealed an ambiguous pattern. The 19 PCR marker products larger than 300 bp, together with the products of 30 markers that produced ambiguous segregation patterns following HRM analysis, were sequenced directly, enabling a further 29 polymorphic markers to be mapped. Thus in total, 94 of the 155 (60.7%) new EST-derived markers were located on the Prunus reference map -derived markers were developed in this study from consensus atabase, ) to 1,26Prunus by Cabrera et al. [Fragaria, Prunus, or in Malus by Vilanova et al. and Sargent et al. [A total of 126 (23%) RosCOS markers bin mapped in a et al.  were loct et al. ,35 were Prunus, and 251 in Fragaria, were used as queries for GMAP and BLASTN. Of these, 1,013 markers  showed a high level of DNA sequence conservation with apple, allowing them to be located to precise positions on the Malus chromosomes using GMAP , 1321 positioned in Malus chromosomes (referred to hereafter as MC) with Prunus and Fragaria was based on the number of reciprocal translocations or fission/fusion events (which we will refer collectively as translocations) and the number of inversions which explain the current marker order in Malus , based on markers in common across the three genera. When the 784 markers shared only between Malus and Prunus were considered, the number of inversions reached 65 across all MC, almost five times that found with the markers in common across the three genera  was also higher than that estimated for the Prunus-Malus comparison. Certain Malus chromosomes, such as MC2, 4, 12 and 15 were highly rearranged between Malus and the two diploid genomes, with two or three translocations per chromosome per species, whereas others  originated each from a single ancestral DNA fragment. The same translocations were found for Malus-Prunus when using markers shared between the three genera or the complete set of anchor markers.The assignment of collinearity of regions of us Table . When coMalus genome, homeologous chromosomes were observed, which corresponded to those identified by Velasco et al. [et al. [Malus chromosomes 3 and 11; 5 and 10; 9 and 17; and 13 and 16. More complex relationships between chromosomes 4, 6, 12 and 14; chromosomes 1, 2 and 7; and chromosomes 2, 8 and 15 were evident. Among genera, eleven major syntenic blocks were common to all three genomes and, in some cases, conservation of synteny was revealed across whole chromosomes. Amongst these, homeologous groups MC13 and MC16 were syntenic to the top of Prunus linkage group (PG)1 and Fragaria linkage group (FG)4, and the top of Malus homeologues MC5 and MC10 corresponded to PG4 and FG3.The locations of markers revealed patterns of conservation of synteny across the genomes of the three genera, as summarized in Figure o et al.  on the b [et al.  on the bFragaria, Malus and Prunus genomes was used to model a hypothetical ancestral genome with nine chromosomes  would have experienced three translocation events, and the Fragaria linkage groups four translocation events, whilst for the Malus chromosomes (x = 17), seven translocation events, two of which  preceded the whole genome duplication in apple. Figure Fragaria, Malus and Prunus showing the positions of major chromosomal translocations necessary to create their extant genomes starting from the putative nine chromosomes of the ancestral genome. Chromosome/linkage group lengths are approximated relative to the lengths of the hypothetical ancestral chromosomes that we assumed to have a similar length. Colouring of the ancestral chromosomes follows Vilanova et al. [A total of 129 markers that mapped to syntenic blocks common to the marker datasets of the three genera were used to develop a model for the ancestral genome of the Rosaceae. The conformation of syntenic blocks shared among the s Figure . Startina et al. .Prunus and Fragaria reference linkage maps and the recently published whole genome sequence of Malus × domestica [Prunus markers meeting the conservation of synteny criteria described in Material and Methods corresponded to 988 Malus loci. The density distribution of these markers was higher than in previous studies involving other families . These values, despite being lower than for Prunus, were still within the range of densities employed for genome comparisons based on linkage maps in species of other families.We have conducted a comparative analysis between sequence-characterised molecular markers genetically anchored to the omestica . In totapersicum ,37-42. TMalus genome sequence. This was expected because RosCOS markers have been developed from genes highly conserved across genomes that are present as low or single copy genes in Arabidopsis [Malus genome. A similar value was also observed for RFLPs generated from cDNA libraries. In contrast, SSRs derived from genomic regions were less conserved among the three genomes, as reported in previous studies [Prunus and Malus compared with Fragaria and Prunus was expected, since Prunus is phylogenetically closer to Malus than to Fragaria [et al. [Fragaria linkage group FG5. The same set of marker data fully support the findings of Velasco et al. [Malus with a genome containing a chromosome complement of x = 9. Our data also revealed a fusion/fission event between A1 and A9 during the formation of MC5 and MC10. This occurred before the major whole genome duplication of Malus and thus was not identified in the analysis of the Malus genome alone.The RosCOS markers showed the highest level of transferability among genomes, with only 4 out of 549 sequences not found in the bidopsis . Transfe studies . While S studies . For exaFragaria . The res [et al.  in an ano et al. , who posMalus implied a wide hybridisation between an ancestral amygdaloid (x = 8) and an ancestral spiraeoid (x = 9) [Malus may have arisen due to a polyploidization of a spiraeoid species [Malus DNA sequence data and that of various Rosaceae species [Gillenia (x = 9) was the closest extant diploid genus to both homeologous genomes of Malus. Our results indicate that from the four pairs of homeologous chromosomes entirely conserved in apple, two pairs, MC3-MC11 and MC5-MC10, were assembled from unique major fragments of two ancestral chromosomes. This suggests that the translocations that produced them pre-date the duplication within the apple genome, and were common to the ancestor(s) of apple. The fact that none of these corresponded to rearrangements found in Prunus indicates that its genome is an unlikely ancestor of apple. In all, these results support the hypothesis that the two genomes that constitute apple were similar, however distant from Prunus, in agreement with the hypothesis of a possible origin of apple as an autotetraploid from a x = 9 genome [Whilst an early hypothesis as to the origin of  (x = 9) , other d species ,47. The  species , and by  species , which s9 genome . Hence, 9 genome  and the Malus and Prunus differed by 19 translocations and 14 inversions (an average of 9.5 translocations and seven inversions per diploid genome) and Malus and Fragaria by 30 translocations and 45 inversions (15 and 22.5). Nine translocations and 27 inversions were estimated when comparing Fragaria and Prunus [Malus and Prunus and more in the comparisons involving Fragaria (36-37.5). Our data suggest that the number of inversions  better estimate the evolutionary distance between genera than the number of translocations, as the comparison of inversions between Fragaria and Prunus yields a smaller number (9) than predicted based on the other two comparisons.Upon pair-wise comparison of each of the three genomes with the ancestral Rosaceae genome Figure , we found Prunus . The tots 15 and 2.5. NinePrunus to the Malus genome, the number of translocations identified was the same as when using the 129 markers in common, however the number of inversions was 65, almost five times higher than that estimated with fewer markers. As expected, most of these inversions were found in relatively small DNA fragments, with 71% (46) of them concerning apple genomic fragments ≤ 7.5 Mbp . Other explanations could account for errors in this estimate, such as wrongly-oriented scaffolds in the whole genome apple sequence, or markers misplaced on the linkage maps. It seems however unlikely that they can account for the majority of the inversions observed. When we re-examined our data and discarded 20 markers that, if misplaced would have generated one or more spurious inversions each, we found a more conservative estimate of 43 inversions (more than three times that of the common set of markers), with 58% of them (25) involving apple DNA fragments ≤ 7.5 Mbp, still a very large number of inversions. We attribute this high frequency of inversions to the fact that our comparison is based on an unusually high average density of markers, allowing the identification of much smaller inverted regions compared with other plant families. Paterson et al. [Sorghum bicolor and Oryza sativa and another comparison between a map of B. napus with high marker density (0.63 markers/cM) with the genome of Arabidopsis (3.67 markers/Mb) allowed the detection of only eight inversions [Drosophila [When we compared the whole set of 784 markers of n et al.  did not versions . A patteosophila , suggestet al. [Based on data from the comparison of genomes of various plant families, Paterson et al.  showed tFragaria, Malus and Prunus to conduct the first detailed family-wide comparative analysis of the Rosaceae. The high density distribution of markers analysed throughout the genomes of the three genera permitted a detailed study of the conservation of synteny, comparable to those performed in other plant families, such as the Solanaceae. Clear syntenic blocks that were conserved across the family were identified, and a hypothetical ancestral genome for the Rosaceae has been reconstructed using 129 common markers. The hypothetical ancestral genome contained nine chromosomes and was comparable to hypotheses proposed in previous studies of Malus, Fragaria and Prunus [Malus and Prunus, based on 784 markers was much higher than that estimated with fewer markers, and higher than any reported genome comparisons in plants, suggesting that small inversions have played an important role in the evolution of these two genera or of the Rosaceae.We have used markers located to the genomes of d Prunus . Our stuPrunus-Malus sequences for a set of genes that were well-distributed throughout the 17 Malus linkage groups, using PRIMER 3 [Prunus reference mapping population (T×E) following Troggio et al. [A total of 155 primer pairs were designed from consensus PRIMER 3  with defo et al. .2, 300 nM forward and reverse primers and 1× HRM master mix (Roche Applied Science). Both PCR and HRM were performed on a Roche LightCycler® 480 (Roche Applied Science). The PCR parameters used were an initial denaturation step of 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 57°C for 15 s, and 72°C for 15 s. Following amplification, the samples were heated to 95°C for 1 min and then cooled to 40°C for 1 min. Melting curves were generated with continuous fluorescence acquisition during a final ramp from 65°C to 95°C at 1.1°C/s and the resultant fluorescence data were processed using the LightCycler480® software .All primer pairs amplifying a single product of 300 bp or less were used to test for polymorphism between the parental genotypes and the six plants of the T×E bin set  using HRet al. [Prunus reference bin map.Amplicons greater than 300 bp in length and those that gave ambiguous results following HRM analysis were sequenced from the parental genotypes and the bin set following the methods of Troggio et al. . SequencM. × domestica genome sequence were screened across the parents of the diploid Fragaria mapping population FV×FN [et al. [et al. [et al. [Primer pairs for the 549 RosCOS markers  that weron FV×FN ,33 follo [et al. . Markers [et al. . Primers [et al. .M. × domestica 'Golden Delicious' [Fragaria and Prunus, to the Malus genome. Marker sequences were used as queries for GMAP [LASTN, using a cut off E-value of 1e-15.The high quality draft genome sequence of licious'  was usedfor GMAP  and as aLAST E-value < e-15) and that were located to the 'Golden Delicious' genome with GMAP, together with the novel EST-derived mapped markers, were used for the conservation of synteny analysis. Syntenic blocks among Malus, Prunus and Fragaria were defined by the following criteria: a syntenic block should contain a minimum of five (Prunus) or three (Fragaria) homologous marker loci mapping to one or two contiguous bins in Prunus and Fragaria, and located within 3.5 Mbp from each other on the Malus genome. Markers originally mapped in the full populations of either Prunus or Fragaria, were converted to bin positions before the analysis. To visualise the relationships between the genomes of the three genera, the Circos software package was used [Malus genome were divided by 4 × 105 to obtain a genetic distance equivalent to marker linkage values of the Malus consensus map [Fragaria and Prunus bin sets, a genetic distance corresponding to the mid-point of each mapping bin was considered. Comparisons between Malus and Prunus, Malus and Fragaria, and Fragaria and Prunus were then visualised, with three linkage groups represented in each ideogram. The method of Vilanova et al. [Markers showing a high degree of DNA sequence conservation in apple . We only included two inversions, one in strawberry and another in apple, that were obvious consequences of the major rearrangements considered in this ancestral genome reconstruction.The set of 129 genetic markers located on the genomes of all three genera were used to define a hypothetical ancestral genome for the Rosaceae. The positions of these markers were used to identify homeologous chromosomal regions within the a et al. , modifieFragaria vesca '815' × Fragaria nubicola '601'; RosCOS: Rosaceous conserved orthologous markers; EST: expressed sequence tag; HRM: high resolution melting; SNP: single nucleotide polymorphisms; MC: Malus chromosome; PG: Prunus linkage group; FG: Fragaria linkage group; WGD: Whole genome duplication.RFLP: restriction fragment length polymorphism; SSR: simple sequence repeat; T×E: almond 'Texas' × peach 'Earlygold'; FV×FN: PA, MT, DJS, DC, SG and RV conceived and designed the experiment. DJS, MT, PA, EI, and DC wrote the paper with editorial comment from the other co-authors. EI, ELG, JB and MP carried out the experiments and provided interpretation of the results. AC and RC performed the bioinformatics analyses and provided interpretation of the results. ACa, EVK and AI gave access to the RosCOS before publication. All authors read and approved the final manuscript.Prunus EST-derived markersTable S1, novel . Table S1 lists locus names, primer sequences, and the T×E bin map positions of the 155 novel EST-derived markers mapped in this work.Click here for fileFragaria genetic markersTable S2, . Table S2 lists the 237 genetic markers of the diploid Fragaria FV×FN reference map, along with their marker type, method of detection and the mapping bins to which they are located.Click here for fileTable S3, markers used in this work. Table S3 lists the number and type of markers used in this work. All the available markers were tested against Malus × domestica genome using GMAP and BLASTN. Syntenic markers are those that fulfilled the criteria defined for conservation of synteny in the Materials and Methods section.Click here for filePrunus and Fragaria map comparisonFigure S1, . Figure S1 shows the comparative analysis between Prunus (PG1 to PG8) and diploid Fragaria (FG1 to FG7) reference map using the Circos program. Only markers that were also present in the Malus genome sequences were included in the Fragaria-Prunus comparison.Click here for fileMalus and Prunus inversions in the Malus genomeFigure S2, . Figure S2 shows the positions in the Malus genome of the inversions detected between Malus and Prunus using the complete set of 784 anchor markers in Prunus. Apple chromosomes are divided in fractions of 10 Mb. Arrows indicate the presence of a predicted inversion.Click here for file


Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK) is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possbile interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT) and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases. Glomerular podocytes are terminally differentiated cells that line the outer aspect of the glomerular basement membrane (GBM). The GBM forms the final barrier against protein loss, which explains why its dysfunction causes protein leakage into the urine, resulting in proteinuria As an important component of the connection between podocytes and the GBM, the reduction of integrin α3β1 might represent one of the mechanisms of podocyturia in glomerular disease. Integrin α3β1 is a membrane glycoprotein consisting of two subunits, a larger α chain and a smaller β chain It is well known that hemodynamic factors, such as blood flow and pressure, exert an important influence on the native structure and function In the present study, the alterations of integrin β1 and FAK in mouse kidneys under various hemodynamic conditions were visualized using IVCT in combination with freeze-substitution, and they were further analysed quantitatively by western blot. Our findings provide new insight into the mechanism of proteinuria during hemodynamic disorders.Rabbit monoclonal (EP695Y) against FAK, mouse monoclonal (P5D2) against integrin β1, and donkey anti-rabbit IgG/TRITC and goat anti-mouse IgG/FITC antibodies were obtained from Abcam . Rabbit polyclonal against FAK phosphotyrosine at 397, biotinylated sheep anti-rabbit IgG and biotinylated rabbit anti-mouse IgG antibodies were purchased from Sigma .Adult C57BL/6J mice, weighing 20–30 g, were used. The experimental animal procedures were approved by the Animal Experimental Committee of China Medical University.The mice were classified into three groups, each containing five mice: a normotensive group; an acute hypertensive group; and a cardiac arrest group. The mice were anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight). In the normotensive group, we resected the left kidney under normal blood circulation. In the acute hypertensive group, we prepared an animal model by ligating the abdominal aorta just below the branching renal arteries for 10 min The renal tissues were then assessed with IVCT, which has been described in previous reports The pieces were freeze-substituted in absolute acetone containing 2% paraformaldehyde (PFA) cooled in dry ice-acetone at −80°C for 48 h and then were gradually rewarmed to room temperature. They were washed in pure acetone twice, transferred into xylene, and then embedded in paraffin wax.As a control group, five kidney specimens were routinely treated with conventional fixation procedures. Some pieces of the tissues were immersed in 4%PFA for 24 h at room temperature, followed by gradual dehydration in a series of alcohols and then transfer into xylene. Finally, the samples were embedded in paraffin wax. The remainder was maintained in −80°C for the biochemical examination.2O2) to block the non-specific reactivity of endogenous peroxidase, followed by a wash with phosphate-buffered saline (PBS). The samples were repaired with sodium citrate buffer liquid at high pressure and were blocked with PBS, containing 5% normal bovine serum  for 1 h at 37°C. Some sections were subsequently incubated with the primary antibodies at 4°C overnight, Other sections were incubated with PBS without the primary antibody. After washing them three times with PBS, the sections were incubated with corresponding secondary antibodies at 37°C for 1 h. Thereafter, they were incubated with horseradish peroxidase (HRP)-conjugated avidin-biotin complex (ABC) for 20 min and were visualized with metal-enhanced 3′3-diaminobenzidine (DAB)  for 5 min (ABC-DAB method). To visualize a clearer structure, we stained them with hematoxylin for 1 min and then dehydrated them in a graded series of alcohols and xylene. Finally, the specimens were sealed with peucine and were photographed under a light microscope or a confocal laser scanning microscope (FV10-ASW2.1 Viewer). Simultaneously, the integral optical density (IOD) of the target protein was measured with Meta Morph , and the result was determined as the sum of the glomeruli. IOD was defined as the sum of the optical densities of all the positive pixels in the image, which represents the quality of the targeted protein.The paraffin-embedded tissues were cut at a 5 μm thickness and were deparaffinized with xylene and a graded series of alcohols. Some of the sections were stained in common hematoxylin and eosin (HE) to observe them morphologically. Other sections were incubated with 3% hydrogen peroxidase . The chemiluminescent signal was captured with a Fujifilm LAS-4000 luminescent image analyzer . Equal protein loading was confirmed by GADPH and Lamin B western staining of the gel. The data shown are representative of at least three independent experiments with similar results.The values are expressed as the means ±SDs. For multiple comparisons with a single control, one-way analysis of variance (ANOVA), followed by Dunnett's test, was employed. The analyses were conducted using SPSS statistical software, version 17.0. P<0.05 was considered to be a statistically significant difference.. In contrast, the capillary loops in sections fixed by immersion were shrunken and there were hardly any erythrocytes in them  continually and evenly under normotensive condition , while uTo visualize the topographical migration of integrin β1 and FAK under hypertensive condition, we detected them using immunoelectron microscopy . In the To further evaluate the expressions of integrin β1 and FAK quantitatively, we examined the two podocyte protein levels in glomeruli. From All of the results were consistent with the immunostaining results and could be attributed to the alteration of renal hemodynamic conditions.It is well known that podocytes are the targets of many forms of injury, and hypertensive injury might be one of the major causes, however, the precise mechanism has not been fully elucidated. In the present study, we visualized the alteration and rearrangement of integrin β1 and FAK under different hemodynamic conditions and as evidenced by IVCT with immunohistochemistry, immunofluorescence and western blot analysis. The results exhibited the reduction and transposition of integrin β1 and FAK under abnormal hemodynamic conditions.It has been generally accepted that kidney morphology and function are dependent on the maintenance of normal blood pressure Under normotensive condition, integrin β1 was continuous and linear-like along the glomerular capillary loop, which is consistent with the previous report Additionally, ischemia and hypoxia are important factors for podocytes. Several studies have documented that ischemia and hypoxia are widely associated with podocytes, except for in tubular and endothelial damage. Mark C. Wagner found that podocytes became swollen, accompanied by narrowed filtration slits, after stopping blood flow for 45 minutes In summary, we visualized the alteration of integrin β1 and FAK during the prophase of the abnormal hymodynamic conditions using IVCT. Integrin β1 and FAK are two essential factors in the regulation of podocyte changes under abnormal hemodynamic conditions. Although the detailed molecular mechanism is not yet clear, pTyr397-FAK is absolutely necessary for the regulation of podocyte changes. Above all, IVCT can maintain all of the substances in situ, providing accurate and clear images. Our next step is to investigate the associated molecules in the integrin-FAK signaling pathway, which should provide new insights into the regulation mechanisms during hemodynamic disorders.Figure S1Confocal laser scanning micrographs showing the co-localization of integrin β1 and FAK on podocytes. In normotensive condition, the micrographs of (A–D) show the localization of integrin β1 (A), FAK (B), and the co-localization of integrin β1 and FAK (D). E, F, G and H present the control stainings ommitting the primary antibodies. The micrographs of (I–L) show the localization of them in hypertensive condition, while (M–P) show the control stainings ommitting the primary antibodies. In the , we detect the pococyte nuclei staining with DAPI. After merging, the overlapping images of Integrinβ1 and FAK was detected, represented by purple color (arrows).(TIF)Click here for additional data file.


Citrus has an extended juvenile phase and trees can take 2–20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees.Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings.The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid. Citrus is grown worldwide, is consumed both fresh and processed, and is one of the most economically important fruit crops. Broadly viewed, new citrus rootstock and scion varieties are developed for three general reasons: 1) improve resistance to pest, disease, and environmental problems to which current commercial rootstock and scion cultivars are susceptible; 2) increase fruit/juice yield to improve profitability to growers; and 3) improve fruit/juice quality to remain competitive in the marketplace. The development of new citrus rootstock and scion varieties takes several decades and can span the careers of multiple researchers. One of the reasons for the long development time is an extended juvenile phase in citrus that typically requires at least 5–10 years before flowering. Another reason is that a new selection is tested at multiple field locations, with multiple rootstocks or scions, and over at least four years of fruit production to provide an estimate of productivity.Plant breeders develop new varieties by introducing genetic variability, generally through controlled hybridizations, into a crop and selecting individuals with useful characteristics. However, there are some serious barriers in citrus biology that must be overcome before progress can be made, and include: the difficulties and expense of working with a tree crop, a long juvenile phase, and many citrus exhibit apomixis and inbreeding depression. These barriers, taken together, make citrus one of the most difficult crops to breed. Genetic engineering is one method to introduce genetic variability into citrus that can potentially address those problems in citrus that have no known viable long-term solution. For example, Huanglongbing (HLB) or citrus greening disease is considered the most serious citrus disease in the world Genetic transformation of citrus rootstock and scion varieties is an active area of research and methods to transform citrus have been reported for many of the major citrus types, including sweet orange Agrobacterium strain, and feeder plates Mature tissue transformation in citrus has been reported for sweet orange 3, GA, basal medium, and growth regulators) for shoot regeneration from mature tissue, a necessary prerequisite for improving the efficiency of mature tissue transformation and, second, to broaden the range of citrus types transformable from mature tissue by including three citrus types not previously transformed including grapefruit, citron, a trifoliate hybrid rootstock, and a high quality and important sweet orange variety  not previously reported.For plant breeding applications, genetic transformation systems should be 1) sufficiently efficient to produce the required numbers of independent transgenics per transgene to alter the phenotype of the targeted trait(s) while retaining the integrity of the variety and, 2) applicable to a broad range of types, commercial and breeding, within the crop. Our objectives were twofold – first, to determine the effects of factors potentially important , C. sinensis (L.) Osbeck Valencia (sweet orange), C. medica (L.) Etrog (citron), and US-942 C. reticulata Sunki×Poncirus trifoliata Flying Dragon) adult phase trees  maintained under greenhouse conditions were used as the source of internode explants . The experiment was repeated and the response at each treatment point was estimated from 9–17 internode explants per replicate. The number of buds/shoots produced was counted and the data analyzed by one-way ANOVA for each citrus type followed by a one-sample t-test that compared the mean of each treatment to 0 .The effect of a dark incubation period on shoot regeneration from internodes of sweet orange, grapefruit, and US-942 was determined. The experiment was designed as a single factor design. Each internode explant was inserted vertically  into a F3 (0 to 5 mg/L), GA (0 to 5 µM), PGR (10 µM ZR or 15 µM BA and 10 µM NAA), and basal medium (MS or WPM). A four-factor response surface design was constructed that included two numeric factors, AgNO3 and GA, and two categorical factors, PGR and basal medium. Design points were selected using D-optimal criterion, modified to include lack-of-fit points, sufficient to estimate a quadratic polynomial. The experimental design included 13 model points, 5 lack-of-fit points, and 5 points to estimate pure error, i.e. 23 design points. The design points are listed in −2 s−1 at 27°C for 21 days, and then data were collected (buds/shoots counted). Data were analyzed by analysis of variance (ANOVA).Four factors considered important or potentially important in shoot regeneration were examined using US-942 and included AgNOAgrobacterium tumefaciens strain EHA-105 strain 600 of 1.3 to 1.5. The Agrobacterium inoculation solution was prepared by diluting the Agrobacterium culture with SIM to a final OD600 of 0.5, and then adding acetosyringone to 40 µM.Agrobacterium inoculation solution for 15 minutes, after which the explants were blotted dry with paper towels. Internode explants were inserted horizontally , incubated in the dark at 27°C for two weeks, transferred to 16/8 h photoperiod, 35 µmol mC. reticulata ’Sunki’ ×Poncirus trifoliata Benecke) and regenerated shoots of US-942 were grafted onto C. volkameriana Ten. & Pasq. (Volkamer).Regenerated shoots >2 mm were grafted onto rootstock seedlings. Valencia, Ruby Red, and Etrog were grafted onto US-812 A rootstock seed was planted in PRO MIX BX soil  in a 164 ml Ray Leach Cone-tainer cell SC10 . PRO MIX BX soil was steamed at 71°C for 2 h 15 min before use. Rootstocks were grown etiolated at 27°C in the greenhouse for a month. When the rootstock seedlings were approximately 10 cm tall, they were used for micro-grafting with transformed shoots. Before grafting, the seedlings were fed with ¼ Basal Salt Mixture  and also given two eye droplets to each pot of a solution of Sequestrene 138, Iron Chelate Mix, 1.2 g/L .GUS assay. Regenerated shoots >2 mm were excised from internode explants and the bottom 1–2 mm of the base of the shoot was excised for assay for GUS activity. The basal sample was placed into a 1.5 microcentrifuge tube containing 200 µL GUS assay buffer Regenerated shoots >2 mm were shaped to look like the alphabet “V” at the base of the shoot. Rootstocks were cut under the cotyledon and sliced vertically in the middle of the stem with a Double Edge Carbon Steel Breakable Razor Blade . Trimmed shoots were inserted into the vertical incision. Immediately after grafting, the Cone-tainer pot was covered with a plastic bag and sealed with a rubber band to retain moisture. Grafted plants were incubated for one week in a growth chamber programmed to a 16/8 photoperiod and 27°C and then transferred to the greenhouse.+ trees and GUS− nontransformed trees using DNase Plant Mini Kits  per the manufacturer’s directions. The uidA gene was amplified with 5′GAATGGTGATTACCGACGAAA3′ and 5′CCAGTCGAGCATCTCTTCAGC3′ designed to amplify a 574 bp fragment DNA was extracted from transformed GUSuidA transgene insertion. The GUS PCR product served as template for the preparation of a DIG-labeled hybridization probe using the DIG DNA Labeling Kit (Roche Applied Science) following the manufacturer’s instructions. All subsequent hybridization, stringency washes, and detection steps were carried out using the DIG detection kit (Roche Applied Science) following the manufacturer’s instructions.Total DNA was extracted from leaf tissue following the method of Dellaporta The effect of a dark incubation period on the number of shoots regenerated was determined for sweet orange, grapefruit, and US-942; citron was not tested. For all three citrus types shoots were only observed with a dark incubation period; no shoots were observed for the 0 week dark treatment. The SIM medium used was based on results from prior experimentation that determined the effects of various cytokinins and auxins on shoot regeneration from juvenile epicotyl explants of sweet orange and grapefruit 3, GA, PGR, and basal medium affected shoot number  and was due to the large positive effect of zeatin riboside. The type of basal medium was also highly significant , though much less so than PGR, with more shoots regenerated on MS medium than WPM. The two-way interaction effects of GA and PGR with basal medium were significant. Quadratic effects  were significant for GA. Based on these results the treatment selected for use in the transformation experiment was MS +5 µM GA +10 µM ZR (replicated design point #9/#23).The number of shoots was counted three weeks after the explants were moved to light. Shoot number for US-942 ranged from 0 to 9.6  and indit number . A summauidA gene fragment and Southern blot analysis using a uidA gene fragment probe . Because these experiments utilized only a 16/8 photoperiod , the effect of a dark incubation period was considered. For juvenile citrus tissue, a dark incubation period enhanced shoot regeneration in sweet orange 3, an ethylene inhibitor, was selected because it can sometimes enhance shoot regeneration 3 was not included in the shoot regeneration medium used for transformation. GA was selected as a potential enhancer of shoot regeneration and growth To further improve shoot regeneration, the individual and combined effects of four additional factors identified as potentially important were determined. The protocol for this experiment included a two week dark incubation period per the results from the dark incubation study. AgNOAgrobacterium-mediated transformation of internode explants from mature greenhouse grown trees of sweet orange, grapefruit, citron, and a trifoliate hybrid rootstock. The results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid. Variability was high and was presumably due to the various relatively uncontrolled environmental and physiological effects. For example, the transformation experiments were conducted over an eight-month period where plant material was harvested from greenhouse-grown trees exposed to light levels and seasonal temperatures that varied widely. Also, the type and condition of the shoot flush can affect regeneration frequencies Transgenic plants were obtained by 3-GA-PGR-Basal medium experiment was conducted only on US-942 and the identified conditions for good shoot regeneration were then applied to all the citrus types. To what extent this effect was due to the inherent responsiveness of trifoliate hybrids vs. using conditions specifically optimized for US-942 is unknown.Transformation efficiency was greatest for US-942. This may be because trifoliate hybrids are some of the most transformable citrus types. However, the AgNO3, GA, type of PGR, and basal medium on shoot regeneration from internode explants from adult phase greenhouse-grown citrus trees. Under our conditions, a dark incubation period was essential and GA, zeatin riboside, and MS basal medium enhanced shoot regeneration. The assumption that treatments that enhanced shoot regeneration would also enhance transformation was made and four diverse citrus were transformed using a single protocol. Because transgenic plants were obtained from these four citrus types, with little control of the initial physiological status of the trees, suggests 1) the potential importance of optimizing protocols to local conditions and, 2) that further improvements in transformation efficiencies across a broad range of citrus types are likely.In this study we have quantified the effects of a dark incubation period and the medium components AgNO


Although iron deficiency is considered to be the main cause of anemia in children worldwide, other contributors to childhood anemia remain little studied in developing countries. We estimated the relative contributions of different factors to anemia in a population-based, cross-sectional survey.12, and folate deficiencies; intestinal parasite infections; glucose-6-phosphate dehydrogenase deficiency; and sickle cell trait carriage. Multiple Poisson regression and adjusted prevalence ratios (aPR) were used to describe associations between anemia and the independent variables.We obtained venous blood samples from 1111 children aged 6 months to 10 years living in the frontier town of Acrelândia, northwest Brazil, to estimate the prevalence of anemia and iron deficiency by measuring hemoglobin, erythrocyte indices, ferritin, soluble transferrin receptor, and C-reactive protein concentrations. Children were simultaneously screened for vitamin A, vitamin B12 , and folate  deficiencies, and C-reactive protein concentrations .The prevalence of anemia, iron deficiency, and iron-deficiency anemia were 13.6%, 45.4%, and 10.3%, respectively. Children whose families were in the highest income quartile, compared with the lowest, had a lower risk of anemia . Child age  and maternal parity  were positively associated with anemia. Other associated correlates were iron deficiency , vitamin BAddressing morbidities and multiple nutritional deficiencies in children and mothers and improving the purchasing power of poorer families are potentially important interventions to reduce the burden of anemia. Iron deficiency (ID) is the most common and widespread nutritional deficiency worldwide. Because global prevalence estimates are not available for ID, anemia is often used as a proxy of ID and iron-deficiency anemia (IDA). Globally, half of the burden of childhood anemia is thought to be caused by ID Recent socioeconomic development, with increased family income, wider access to education, and better income distribution, has led to a major decrease in the prevalence of child stunting in Brazil The prevalence of childhood anemia due to ID in Brazil remains largely unknown because hemoglobin (Hb) measurements used in nationwide representative surveys are neither sensitive nor specific as a screening test for ID. Lack of sensitivity occurs because a large proportion of total body iron must be lost before Hb concentrations fall below the conventional cut-off points to define anemia 12, and folate deficiencies; intestinal parasite infections; G6PD deficiency; and sickle cell trait carriage. The aim of the study was to estimate the relative contribution of ID and other nutritional and non-nutritional factors to anemia in this population to provide a basis for public health intervention.In the present investigation, Hb, PF, sTfR, and CRP measurements were combined to determine the iron status of Amazonian children between 6 months and 10 years of age. In this population-based, cross-sectional study, children were simultaneously screened for vitamin A, vitamin BThis study protocol was approved by the institutional review board of the School of Public Health, University of São Paulo, Brazil (No. 1681/07). Written informed consent was obtained from the parents or guardians of children who participated in the study. Data were analyzed anonymously.A population-based, cross-sectional study of children’s nutrition and health was carried out in December 2007 in Acrelândia, a frontier town situated 112 km east of Rio Branco, the capital of the state of Acre, western Brazilian Amazonia. The adult inhabitants of Acrelândia  are mainly migrants from southeast and south Brazil who are currently engaged in commercial agriculture and cattle raising. Infant mortality in Acrelândia, estimated at 70.7 per thousand live births in 2000, is substantially higher than the country average (27.7 per thousand live births) Sampling strategies, questionnaires, and field procedures were similar to those of a previous survey carried out in the same area in 2003 Length/height and weight were measured during the clinic visit by trained research assistants following standardized procedures and using calibrated equipment 12 concentrations were measured using commercial fluoroimmunoassays ; values <10 nmol/L and <150 pmol/L were considered diagnostic of folate and vitamin B12 deficiency, respectively A sample (approximately 5 mL) of fasting venous blood was collected from 1111 children aged between 6 months and 10 years (90.7% of those enrolled). Because limited sample volumes were available, not all laboratory analyses were performed for all children. At the field laboratory in Acrelândia, whole blood aliquots collected in EDTA-containing vacuum tubes were used to carry out full blood cell counts and measure Hb concentrations on an automated cell counter . A separate blood sample was protected from light and centrifuged within 1 h of collection; serum and plasma samples were shipped to São Paulo on dry ice and frozen at –70°C until further analysis. In São Paulo, PF and sTfR concentrations were measured using commercially available enzyme immunoassays ; CRP concentrations were measured using a high-sensitivity chemiluminescent assay . Anemia, ID, and IDA were defined according to Hb, PF, sTfR, and CRP concentrations as follows DNA-based tests were used to screen for G6PD deficiency and sickle cell trait carriage. DNA was extracted from EDTA-containing whole blood samples using DNA kits . The G6PD202A mutation (rs1050828) was genotyped, constituting the most frequent diagnostic single-nucleotide polymorphism associated with G6PD deficiency found in the Amazon Basin of Brazil Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. Children with intestinal parasitic infections received free treatment prescribed by research clinicians.Stool samples were collected in plastic containers containing a preservative solution  from 989 children aged between 6 months and 10 years (80.7% of those enrolled). Logistic limitations prevented the collection of more than one stool sample from each participant, but prevalence estimates derived from examination of a single sample have been shown to be accurate for most infections with soil-transmitted helminths (geohelminths) associated with anemia P<0.10 and for ordinal variables when they followed a dose-response pattern or if their inclusion in the model changed the PR by 10% or more. Missing observations were included in the multiple models by creating missing-value categories. Potential correlations among siblings within the same household were assessed in multilevel regression models using the STATA “xtmepoisson" procedure. Estimates of the population attributable fraction for dichotomous exposures were calculated using the following formula: attributable fraction = p (PR-1)/PR, where p is the proportion of cases exposed, and PR is the adjusted PR Principal component analysis was used to derive a wealth index, a proxy of household income Of the 1151 participants, 1111 children (97%) >6 months of age provided blood samples and constituted the analytic sample. On average, there were 1.7 participants per household, ranging from 1 to 5 children. No sanitation system was available in the town. Among those identified as either head of the household or the primary caregiver, 38.1% had studied for <5 years. The race/ethnicity of the majority of the participants was classified as brown (86%); a high proportion of mothers with only primary school education (38%) and a history of more than two pregnancies (64%) was observed .12, and folate were 5.5%, 14.2%, 3.7%, and 2.5%, respectively. Only one case of sickle cell disease was observed. Of the 1034 children for whom plasma CRP information was available, only 41 (4%) had CRP values of ≥10 mg/L. The CRP index yielded similar percentage estimates of ID in all age groups.Median values and interquartile ranges for iron status indicators and prevalence of anemia and other conditions are shown in P<0.05, χ2 test). Microcytosis  compared with preschool and school children . Attributable fractions were estimated for factors other than age, sex, wealth index, maternal schooling, and maternal age  derived from multiple Poisson regression models with anemia as the outcome. Children living in families that belonged to a higher wealth index quartile were at lower risk for anemia . In contrast, children <2 years of age  and children whose mothers had more than two pregnancies  were at higher risk for anemia. Other associated risk factors were ID, deficiency of folate or vitamin Brnal age . Overallrnal age . Similar12, and CRP were strong independent predictors of Hb concentrations. The final model explained 37.6% of the variance of Hb concentration in the children studied. On average, (a) increases of 1 mg/L of sTfR and 2.72 mg/L of CRP (1 log unit) were associated with 1.21 and 0.31 g/L decreases in Hb, respectively; (b) decreases of 1 µmol/L of retinol and 1 pmol/L of vitamin B12 were associated with 1.71 and 0.01 g/L decreases in Hb, respectively (P = 0.000).In addition to age and wealth index, circulating concentrations of sTfR, retinol, vitamin Bectively . In sepa12 and folate and high plasma CRP (approximately 3% for each risk factor).Anemia in the Amazonian children studied was twice as prevalent as that in comparable population groups from industrialized countries 12 and folate deficiency found in this study  was low, vitamin B12 deficiency was observed in 12% of examined children under the age of 2 years and was associated with anemia. Comparisons with available data within and between countries are difficult because the majority of data on the prevalence of folate and vitamin B12 deficiencies are derived from relatively small surveys with different nutritional status indicators. Anemia due to folate and vitamin B12 deficiency has been considered uncommon worldwide. This is probably due to limitations in diagnosis because iron deficiency tends to lower the mean corpuscular volume (MCV) in the complete blood count more readily than it is raised by folate or vitamin B12 deficiency while the use of other biochemical indicators is expensive 12 deficiency (<148 pmol/L) in 1.6% of children. Plasma vitamin B12 concentrations were inversely related to the mother’s parity and positively associated with socioeconomic status and a dietary pattern that included frequent intake of beef, chicken, and dairy products in a dose-response manner 12 deficiency alone (<150 pmol/L) and 66% had at least one micronutrient deficiency  12 deficiency among toddlers in the present study might be related to low daily intakes of vitamin B12 as described in a previous study in the Amazon area Although the overall prevalence of vitamin BThe high prevalence (14.2%) of vitamin A deficiency observed in our population of a similar magnitude across all age groups is of particular concern. Although this vitamin A deficiency was not found to be associated with anemia, a positive association between serum retinol and Hb concentration was observed, as reported by other authors in studies of children in Latin America and elsewhere As expected, the high frequency of morbidities observed in this study population increased the concentrations of both PF and sTfR Previous studies have demonstrated that the role of anemia of inflammation in the pathogenesis of anemia within the developing world plays a central role in the context of infectious diseases prevalent in these regions A body of evidence reinforces the concept that anemia is a marker of socioeconomic disadvantage, with the poorest and least educated being at greatest risk of exposure to risk factors for anemia and its sequelae In our study, high maternal parity (more than 2 pregnancies) was positively associated with anemia. In developing countries this factor can be considered as a marker of early onset of childbearing, short intervals between births, and poor access to antenatal care and supplementation Certain limitations in the present study should be noted. The principal constraint of our study is its cross-sectional design. Therefore, the temporal relation between exposures and outcomes (anemia or Hb concentrations) could not be established to ensure that micronutrient deficiencies and elevations in CRP concentrations precede the outcomes. Despite this limitation, we have identified a comprehensive set of associated factors with anemia and Hb concentrations in Amazonian children aged 6 months to 10 years. No other population-based surveys of anemia and ID in Brazil have used a wide-ranging combination of biochemical indicators; available national estimates of ID prevalence have mostly been based on Hb determination.12 and retinol, and elevated CRP concentrations as a measure of recent infection were additional factors shown to affect Hb concentrations. Strategies to control anemia based exclusively on iron supplementation are likely to have only a limited impact on the overall prevalence of anemia. Controlling morbidities and multiple nutritional deficiencies and improving maternal nutritional status and the purchasing power of poorer families represent potentially important interventions.In conclusion, approximately 5% of all cases of anemia in our study population were attributable to ID; a similar attributable fraction was estimated for high parity (more than two pregnancies) among mothers. Among infants and toddlers, almost one in five cases of anemia was attributable to ID; purchasing power, serum concentrations of vitamin B