Pelecanus erythrorhynchos), common loon (Gavia immer), and northern gannet (Morus bassanus). Point counts  or strip transects (gannet) were used and each target species was assessed for oiling  and photographed. Due to distance at sighting and/or poor visibility, not all visible birds were assessed. The percentage of birds oiled varied by species, with the common loon being the highest (23.6%), followed by American white pelican (16.9%), and northern gannet (6.9%). Most of the American white pelicans and common loons had trace  or light levels  of oiling. The northern gannet had just trace levels of oiling. Some pelicans (6%) and loons (4%) had moderate amounts of oiling. Based on expert derived-mortality estimates and our estimates of oil exposure, we used Monte Carlo simulations to predict expected decreases of 2.5%, 4%, and 11% in the observed population for the northern gannet, American white pelican, and common loon, respectively. While these values are underestimates of the true values given the long time lag (10–12 months) between the oil spill and the assessment, these data represent some of the few estimates of exposure for these species and describe minimum risk estimates to these species.The Natural Resource Damage Assessment and Restoration Trustees for the Deepwater Horizon oil spill assessed the external oiling of migratory bird species dependent on open water in the Gulf of Mexico following the aforementioned spill. The assessment was designed to evaluate birds that use open water during the winter within 40 km of the Gulf shoreline. We focused on the American white pelican ( Seabirds are excellent indicators of oil pollution because they spend most of their time in marine environments on the ocean surface where oil tends to disperse and persist after spills , which tends to inhabit coastal waters (King and Michot Gavia immer), which tends to inhabit nearshore waters (Jodice Morus bassanus), which inhabits both nearshore and offshore waters. These target species all have deferred maturity, high adult survival rates, and low reproductive rates, so any impacts that lower adult survival could have long-lasting population level effects (Furness and Monaghan As part of the avian injury assessment for the Deepwater Horizon (DWH) oil blowout, the US Fish and Wildlife Service, together with the Trustees, assessed the oiling of migrant bird species on the open water in the first winter (2011) following the spill. The DWH spill occurred roughly 80 km off the Louisiana shore and the surface oil covered > 175,000 kms Jodice ; and theThe study area ranged from Terrebonne Bay, Louisiana (LA), to Apalachicola, Florida FL; Fig. , as thisThe entire study area, from coastline to 5 km out at sea was organized by Area Contingency Plan (ACP) grid cells as adopted by the LA Coast Guard Fig. . The pelPelican and loon surveys were conducted primarily using boat-based observations (21 ft bay boat) within nearshore habitats, such as bays and coves, as well as along barrier islands, primarily in LA and Mississippi (MS); a few surveys were also conducted from shore. Once biologists entered a specific ACP grid, they immediately looked for target species and if any were observed, approached them (to within 30–70 m) to visually assess each individual for oiling. To assess oiling rates, observers used either 8 × 40 or 10 × 50 binoculars from boats or spotting scopes (20–60×) from land. Every attempt was made to photo document all birds assessed and the level of oiling. Pelican and loon surveys were conducted from 17 February to 15 April 2011 and 17 February to 15 March 2011, respectively.Northern Gannets were assessed using systematic boat-based strip transects. Transects were spaced every 10 to 20 km, beginning from shore and extending out to 40-km offshore. They ran either perpendicular to shore, parallel to a selected set of grid lines, or used a hybrid approach , observers determined, based on the gannet’s general behavior, whether the aggregation was expected to remain in place long enough to allow for assessment. If so, strip transects were paused so the vessel could move off transect and approach the aggregations and conduct a point count. Once that point count was completed, the vessel returned to transect and continued the strip-transect methods. Observations were conducted from an elevated viewing platform that provided an unobstructed view of the water. Gannets and other seabirds within a 90°, bow-beam arc and within 300 m of one side of the ship were recorded, following standard at-sea survey protocols , “Light” (6–20%), “Moderate” (21–40%), and “Heavy” (> 40%). Oiling rates were only estimated for birds when more than 50% of the body surface could be clearly observed. Each bird that was clearly observed was assigned an oil exposure category based on visual and photographic inspection. A laser range finder was used to measure the distance between the observer and birds observed; however, measurements were not made for each individual bird if found in a group. Observations were geo-referenced using GPS coordinates.During surveys, bird oiling levels were categorized as “None,” “Trace”  to determine how much of a decrease in target species populations would have been expected based on the estimates of oil exposure. For each iteration of the simulation, we began with the total number of detections of each target species then assigned a subset of the total to oil exposure categories at the exposure rates documented in this study using a random binomial draw. For each species, we then estimated the probability of mortality at each exposure level using the Trustee expert panel data. Mortality estimates were provided as ranges rather than estimates of central tendency with a variance estimate, so we used a uniform distribution to randomly select any value within the range for each simulation. Last, we used another binomial draw to determine the number of birds that survived their oil exposure in each exposure category based on the total number of birds in that exposure category and the randomly selected mortality probability. We ran these simulations for 100,000 iterations (the point at which the results appeared to be properly stable across multiple seeded attempts) for each species using the R statistical computing environment  or light  extents, and some with moderate oiling levels  in LA, 10 (2.1%) in MS, and 77 (15.9%) in AL. During the surveys, 2407 American white pelicans were observed and most met the criteria for oil exposure assessment . Visibly oiled loons were observed in 11 ACP grid cells . The distribution of oiled common loons, however, was not uniform across ACP grid cells or states, with most oiled birds being recorded in LA.Forty-five loon surveys were carried out, 20 (44.4%) in LA, 11 (24.4%) in MS, seven (15.6%) in Al, and seven (15.6%) in FL (Table n = 81), while 25 (23.6%) showed visible oiling. Of these, 18 had trace levels (72.0%), six had light levels (24.0%), and one showed moderate oiling , followed by AL , FL , and MS . Given the level of oiling and the corresponding mortality estimates, the MCS predicted a population decrease of 10.7% (95% CI 6.0–15.8%).Field observers tallied 1148 common loons. Following the study protocol, however, only loons that were clearly visible (> 50% of body surface) were assessed. Thus, a total of 106 (9.2%) common loons were assessed for oiling. Most did not show visible oiling  were conducted opportunistically when aggregations of northern gannets and other birds occurred within sight of observers from their transect position.A total of 36 survey transects were completed, and ~ 30% of two additional transects were completed. In total, more than 1400 transect km were surveyed Fig. . Point cn = 83) were not visibly oiled and a few  showed trace levels of oiling. After reviewing the photographs, two additional birds were determined to be visibly oiled, making the oiling rate of northern gannets 6.9%  of them observed clearly enough to be visually assessed for oiling Table . Of the 9% Table . Given tObservations of oiling rates for American white pelicans, common loons, and northern gannets were made in February–March 2011, almost a year after the Deepwater Horizon oil spill originated, and 7 months after the well was capped and new oil stopped entering the system. By this time, much of the surface oil from the spill was removed by a variety of remediation responses , had evaporated, reached land, or settled on the sea floor sediment . These factors, in addition to their preference for foraging in the shallows where tar balls and other oil could accumulate , these surveys occurred 9 months post-blowout and individuals that were previously in the moderate to heavy oiling category may have perished, representing acute mortality, and were removed from the population prior to surveying. Even birds with trace to light oiling levels can experience mortality Ziccardi . The cumOiling is just one of many stressors these target species experience on their wintering grounds. Cleanup activities following the spill were ongoing during the sampling period and the additional human disturbance was found to have impacted the fattening rate of shorebirds , our simulation work provided some context for the population implications of the exposure rates documented in this study. Of the three target species, based on the mortality estimates decided on by the expert panel for each species Ziccardi  and the Thousands of American white pelicans, common loons, and northern gannets were observed in the northern Gulf of Mexico during the winter following the Deepwater Horizon spill. Despite being 7–11 months after the origin of the blowout, the oiling rates of these waterbirds ranged from at least 7 to 24% and these oiling rates likely caused significant mortality in these populations (1–11% above unoiled populations). Categories of oiling were most often trace, though both light and moderate oiling were also observed. Any American white pelicans, common loons, and northern gannets that were moderately  to heavily oiled  had probably died by the time the surveys were being conducted and may explain why so few birds were observed in these oiling level categories. Birds with trace, light, or moderate amounts of oiling both inhaled and ingested the oil, and chronic exposure to petroleum can have numerous sublethal effects, which, depending on the concentrations of the oil and duration of exposure, may ultimately have population-level effects Albers . The pot


Cholestasis is a common but serious clinical condition in preterm neonates. The current management for preterm neonatal cholestasis has limitations. The aim of this study was to determine effects of Bifidobacterium supplementation on the prevention and alleviation of cholestasis in preterm infants with very low birth weight.  Preterm neonates with very low birth weight were enrolled in the Children's Hospital of Soochow University between December 2012 and December 2017. The patients were randomly assigned into Bifidobacterium and control groups, and effects of Bifidobacterium supplementation on the outcomes were compared between the two groups. P < 0.01). Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests (P < 0.01). Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests (P < 0.01). Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests . Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests (P < 0.01). Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests (P < 0.01). Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests (P < 0.01). Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests (P < 0.01). Furthermore, the Bifidobacterium group exhibited less severe cholestasis and better improvement of the liver function than the control group as evidenced by the biochemical tests . Pretermlestasis –6.The current recommendations for the management of the preterm neonatal cholestasis, such as conservative phototherapy and surgical intervention, have limitations, and there are a number of concerns over adverse effects. Therefore, prevention and alternative treatments would be valuable in this regard. In addition, there is a need for an improvement and better care for cholestasis in preterm neonates and those with cholestasis. Notably, the gastrointestinal function in newborns especially preterm infants is still immature, for which parenteral nutrition (PN) is required. The implementation of PN and delay in the enteral feeding may cause PN-associated cholestasis (PNAC) or even make the existing cholestasis worse. Long-term PN can also result in an imbalance in the intestinal flora, damage of the gastrointestinal mucous, and disruption in the bile secretion and further aggravate the conditions of cholestasis . TherefoIn this study, we aimed to assess effects of prebiotic supplementation on the potential prevention and alleviation of severity of neonatal cholestasis in preterm neonate with very low birth weight. The findings obtained through conducting this study may provide scientific evidence of the prebiotic supplementation in the better management of preterm newborns with very low birth weight.In this prospective, double-blinded, randomized comparative study, 510 preterm neonates in the Children's Hospital of Soochow University between December 2012 and December 2017 were screened for their eligibility. The following inclusion criteria were used: (1) admission to the hospital within 12 hours after birth, (2) gestational ages ranging from 28 to 34 weeks, (3) very low birth weight (<1500 g), and (4) total parenteral nutrition (TPN) one day following birth. However, the patients who presented the following conditions were excluded from this study: (1) congenital heart diseases, malformations in the gastrointestinal (GI) tract, and genetic metabolic diseases; (2) surgical treatment during hospitalization; (3) severe symptoms of digestive diseases  before TPN; (4) cholestasis at the time of admission to our hospital; (5) taking gastrointestinal stimulants; (6) taking antibiotics; and (7) incompletion or withdrawal of treatment during hospitalization. Ten patients were excluded from this study, including one congenital birth defect, five severe symptoms of digestive diseases before TPN, and four cholestasis diagnosed at the time of admission. As a result, a total of 500 study subjects were enrolled in this study.All the guardians/parents of the infants had given written informed consent prior to enrollment in the study. This study was reviewed and approved by the Ethics Committee of the Children's Hospital of Soochow University .Lactobacillus acidophilus, and Enterococcus faecalis at no less than 1.0 × 107 cfu/g [All the study preterm neonates were randomly assigned into the Bifidobacterium group and the control group using the stratified permuted block randomization. The patients in the Bifidobacterium group were given orally Bifidobacterium triple viable powder  within 24 hours after birth until TPN was implemented, while those neonates in the control group were orally fed without Bifidobacterium supplementation. Bifidobacterium triple viable powder contains live bacteria of long Bifidobacterium, 07 cfu/g , 15.Before having reached enteral feeding, PN was implemented to prevent hypoglycemia, hyperglycemia, and electrolyte acid-base balance disorders. In addition to routine hepatoprotective therapy , ursodeoxycholic acid  combined with jaundice Yinchen granules  was used for the treatment of cholestasis.Both groups were fed with formula milk powder designed for preterm infants (osmotic pressure 327 mOsm/L) with an initial 3-5 mL/(kg·d), less than 20 mL/(kg·d). The daily milk quantity and calorie of each infant were monitored to determine at least 90-120 kcal/(kg·d) .The formula of PN includes 20% medium-long chain fat emulsion, water-soluble vitamins, fat-soluble vitamins, amino acids essential for infants, glucose, electrolytes, and trace elements. The initial dosage of amino acid was 1.5-2.0 g/(kg·d), then gradually increased to 3.5-4.0 g/(kg·d); the initial dosage of fat milk was 0.5-2.0 g/(kg·d), which gradually increased to 2.0-3.0 g/(kg·d); and the dosage of glucose gradually increased from 4-6 mg/(kg·min) to 11-14 mg/(kg·min). PN was administered through a peripheral venous micropump for 8-12 hours.μmol/L (5 mg/dL), conjugated bilirubin > 17:1 μmol/L (1 mg/dL) or serum TB > 85 μmol/L, and conjugated bilirubin/total bilirubin > 20%. Biochemical tests for the liver function were performed every 5 days in the study subjects. Cholestatic liver damage was diagnosed based on the results of blood tests indicating that the serum bilirubin, alkaline phosphatase, gamma glutamine transferase, ALT, and AST level were increased when the child experienced jaundice, hepatomegaly, anorexia, dark urine color, light stool color, and other clinical symptoms. Necrotizing enterocolitis (NEC) in newborns was diagnosed based on the clinical presentation of symptoms such as bloody stool, abdominal distention, vomiting, lethargy, apnea, and hypotonia and if abdominal X-ray examination showed cystic emphysema of the intestinal wall. However, atypical clinical manifestations required differentiation from other diseases. Extrauterine growth retardation (EUGR) was diagnosed if the weight, length, or head circumference of the child was below the 10th percentile of the expected value for the corresponding intrauterine growth rate at the time of discharge. The clinical manifestations of septicemia were nonspecific, mainly including poor response, fever or lack of temperature rise, lack of weight increase or slow growth, jaundice, hepatosplenomegaly, shock, and even multiple organ lesions. The results of nonspecific examination included increased or decreased peripheral blood leukocyte count, a ratio of baculonuclear cells to neutrophils of ≥0.16, a platelet count of <100 × 109/L, increased C-reactive protein level, increased serum procalcitonin level, and increased IL-6 level. Septicemia was diagnosed based on clinical manifestations and confirmation of any of the following conditions: (1) pathogenic bacteria identified in blood culture or sterile coelomic fluid culture and (2) if opportunistic pathogens were found in blood culture, the same bacteria were also found in another blood culture, catheter tip, or sterile body cavity. Presentation of clinical manifestations and any of the following conditions resulted in a clinical diagnosis of septicemia: (1) at least two abnormal nonspecific examination results and (2) positive result on pathogen antigen or DNA testing. All diagnostic criteria were obtained from literature reports of studies conducted outside of China [The levels of TB, direct bilirubin (DB), gamma glutamine transferase, total bile acid (TBA), and total cholesterol (TC) in the infant patients with cholestasis were recorded. The diagnosis of cholestasis was primarily made according to the following biochemical examinations: TB < 85 of China –13, 17.t-test was used to compare the means of two sets of data. For data without satisfying the normal distribution, they were expressed as the median value ± interquartile range (IQR), and the two groups were compared by a nonparametric rank sum test that analysed the difference. The Chi-squared (χ2) test was used for comparison of enumeration data of two paired groups. P < 0.05 was considered as significant difference in all statistical tests.SPSS17.0 statistical software was used for the statistical analysis in this study. The data with normal distribution were expressed as the mean ± standard deviation (SD), and the independent sample A total of 511 preterm neonates were evaluated for eligibility, of which 500 infants were finally enrolled in this study. The study patients were randomly assigned into two groups with 250 neonates in the control group and in the Bifidobacterium supplementation group. The baseline demographic and clinical characteristics of the study patients, including methods of childbirth, Apgar scores, gestational age, and gender, were summarized in Suppl. P < 0.01). Additional analysis of neonatal cholestasis-associated complications showed that the Bifidobacterium group exhibited significantly lower proportion of cases that had the following cholestasis-associated complications compared with the control group: cholestatic liver injury , NEC , FI , and EUGR (8.4% versus 14.8%). It was of note that a proportion of septicemia was slightly lower in the neonates with Bifidobacterium supplementation compared with those with placebo, whereas the difference between the two groups was not statistically different .To determine a potentially preventive effect of Bifidobacterium supplementation on neonatal cholestasis, we compared incidence rates of neonatal cholestasis and its associated complications in the two groups with or without Bifidobacterium supplementation. As presented in μmol/L, P < 0.05), peak direct bilirubin , γ-glutamine transferase , alkaline phosphatase , total bile acid , and total cholesterol .We also examined effect of Bifidobacterium supplementation on the severity of neonatal cholestasis in the study infants, which were assessed on the basis of a range of biochemical tests for the disease severity. As shown in P < 0.05).We also compared effect of Bifidobacterium supplementation on the improvement of the liver function in the study infants based upon the laboratory tests for the liver function. As detailed in P < 0.01). The Bifidobacterium group exhibited significantly better clinical outcomes in comparison with those in the control group (P < 0.05) over the period of this study as evidenced as follows: days required for the study neonates to meet the full enteral feeding were significantly fewer in the Bifidobacterium group than those in the control group , duration of meconium passage was significantly shorter in the Bifidobacterium group than that in the control group , proportion of cases on fasting and duration of fasting were significantly lower in the Bifidobacterium group than those in the control group , and duration of weight gain to normal was significantly shorter in the Bifidobacterium group than those in the control group .Finally, we made comparison of the duration of hospitalization and other clinical outcomes between the two groups with or without Bifidobacterium supplementation, and the main findings were summarized in This study of effects of Bifidobacterium supplementation on cholestasis in preterm infants with very low birth weight has the following main novel findings: (1) Bifidobacterium supplementation significantly reduced the risk for neonatal cholestasis and its related complications in the preterm infants with very low birth weight ; 2) Bif Bif2 andTo our knowledge, the studies of efficacy of Bifidobacterium administration on cholestasis in the preterm infants with very low birth weight are limited. Many previous studies focused on evaluation of impacts of prebiotics on the care for preterm neonates, and benefits of prebiotic administration included improvement of the stool characteristics, reduction of the enteral feeding tolerance, and increase in the gastrointestinal motility , 18–22. 7 cfu/g [Bifidobacterium has been detected in fecal samples of infants at the ages of 3–276 days and is among the main beneficial bacteria with a minimum concentration of 1.0 × 107 cfu/g . Accordi7 cfu/g . We alsoLactobacillus acidophilus and Enterococcus faecalis. Although these probiotics have been reported to have little impact on human health [Our study may have a few potential limitations. First, the duration of intervention was relatively not long, for which the long-term safety and efficacy of Bifidobacterium supplementation on the preterm infants with very low birth weight are unknown. Second, Bifidobacterium trifecta contains small amounts of n health , it is nTaken together, we have found the beneficial effects of Bifidobacterium supplementation for the management of cholestasis in the preterm newborns with very low birth weight, possibly through improving the colonization of the beneficial intestinal bacteria and the enteral feeding tolerance, as well as decreasing the enterohepatic circulation of bilirubin. The long-term safety and effectiveness of Bifidobacterium supplementation will need further clinical investigation with longer duration of intervention in a large sample size.


Tumoral calcinosis (TC) is an uncommon disease that has been linked to familial genetic mutations but can often be due to secondary causes such as chronic renal failure and hyperparathyroidism. There are rare instances of tumoral calcinosis induced by foreign body injections, often for cosmetic purposes. Here we describe operative management of spinal cord compression due to mineral oil injection induced tumoral calcinosis. A 54-year-old transgender female presented with signs of myelopathy so severe that she had become wheelchair bound. Labs demonstrated hypercalcemia and imaging of the neuroaxis revealed significant calcification resulting in cervicothoracic and lumbar central canal stenosis. Given symptomatic cervical spinal cord compression, she was taken to the OR for urgent laminectomy and decompression. Postoperatively, she recovered well and was ambulating independently by postoperative day (POD) 9. This is the first reported case of localized mineral oil injections causing distant calcification with subsequent symptomatic cord compression requiring operative intervention. Tumoral calcinosis (TC) was first described in 1948, and since then a number of underlying etiologies have been discovered -4. The tHistory A 54-year-old transgender female presented to the ED with five months of progressive gait instability resulting in debilitation so severe that she had become wheelchair dependent. She also reported lumbar back pain and right lower extremity radiculopathy. Her history was notable for HIV+ intermittently compliant on antiretroviral medications, hypercalcemia, and chronic kidney disease. She also had undergone two body contouring procedures with mineral oil injections of her buttocks and thighs 20 years prior in the process of transitioning to a woman. Physical exam revealed right lower extremity paresis. In her right lower extremity she had 3/5 strength in dorsiflexion and 4/5 throughout the rest of the leg. There was decreased sensation in bilateral lower extremities with 4+ patellar deep tendon reflexes and ankle clonus. Her buttocks and posterior thighs demonstrated diffusely hard darkened skin, and a 10 cm x 10 cm well-circumscribed midline nodule at the level of the sacrum. A separate but similar 5 cm x 5 cm nodule was also noted in the lumbar area. Laboratory results demonstrated hypercalcemia (13.2 mg/dL), depressed parathyroid hormone (PTH) (4.8 pg/mL), slightly elevated parathyroid hormone-related protein (PTHrp) (5.0 pmol), and elevated Vitamin D 1,25 OH (248 pg/mL).The MRI of the neuroaxis demonstrated a small amount of posterior epidural fluid from C7-T3, multilevel degenerative changes of the cervical spine, and severe spinal canal stenosis from C7-T1 resulting in cord compression and myelomalacia Figure ,B. CT ofOperative procedureShe was placed prone on a Jackson table in Mayfield pins. A midline cervicothoracic incision was made, and the posterior elements exposed. A standard midline laminectomy was performed, removing the entire lamina of C7 and T1, with extension to the inferior portion of C6, and superior portion of T2. The lamina was adherent to an epidural collection of calcified phlegmon which was adherent to the dura. This was carefully excised using a combination of curettes and Kerrison rongeurs until normal appearing dura was visualized at the margins of the laminectomy, and the spinal cord adequately decompressed. The phlegmon was sent to pathology and microbiology. The wound was irrigated, hemostasis achieved, and closed in layers with a drain left in place. Postoperative courseImmediately after surgery, she demonstrated significant improvement in right lower extremity strength, numbness, and gait. By postoperative day (POD 9) she regained full strength with significant improvement in deep tendon reflexes and clonus. In addition to cervical decompression, she also underwent a lymph node biopsy. Final spinal pathology demonstrated fibrous connective tissue with nonpolarizable calcifying crystalline material associated with foreign body-type granulomatous reaction, and calcification consistent with TC Figure . The lymTumoral calcinosis is an uncommon entity, and a rare finding in the spine. The largest series describes 21 cases of nearly missed diagnosis due to the variable presentation on MRI, CT, and XR . Familia Nonparathyroid hypercalcemia has been recently linked to exogenous foreign body injections with silicone, paraffin oil, and mineral oil which may be used during cosmetic procedures as dermal fillers for body contouring , 4. Loca While hypercalcemia and TC as a reaction to cosmetic injections has been linked to local inflammatory and calcific reactions, this is the first report of symptomatic TC of the spine due to physically distant cosmetic mineral oil injections. Other causes of this calcification process include infectious and malignant etiologies, neither of which were likely causes in our patient. Most likely, the body contouring mineral oil injections induced a granulomatous reaction causing systemic hypercalcemia and subsequent TC. This is supported by abnormally high levels of Vitamin D 1,25 OH, moderate hypercalcemia, and low PTH. The mechanisms that predispose the spinal epidural space to the development of TC in our patient are obscure. One possibility is retrograde lymphatic spread of the local mineral oil injections to the epidural space, altering the intracellular lipid content and subsequent abnormal response to hypercalcemia. It is also possible that lymphatic spread of the mineral oil directly induced kidney damage. We present the first reported case of compressive TC in the cervical spine due to cosmetic injections of mineral oil causing granulomatous reaction and hypercalcemia. A thorough history, extensive medical workup, and careful evaluation of imaging are key elements in obtaining an accurate diagnosis to optimize the outcome of these rare cases.


Chlorella vulgaris and subsequent uptake by wheat (Triticum aestivum L.) . Absolute and relative leaf area growth and estimated P uptake rates showed similar dynamics, indicating that wheat acquires P from algae quickly. A model proposes that algal fertilizer promotes wheat growth after rapid transformation in soil to inorganic nutrients. We conclude theoretically that phosphorus from algal biomass is available to wheat seedlings upon its application and is released gradually over time with minor differences related to moisture content on application. The growth and P uptake kinetics hint at nutrient forms, including N, and biomass stimulation worthy of research to further exploit algae in sustainable agriculture practices. Temporal resolved phenotype analyses in combination with a mass-balance approach is helpful for understanding resource uptake from recycled and biofertilizer sources by plants.Algae can efficiently take up excess nutrients from waterways, making them a valuable resource potentially capable of replacing synthesized and mined fertilizers for agriculture. The capacity of algae to fertilize crops has been quantified, but it is not known how the algae-derived nutrients become available to plants. We aimed to address this question: what are the temporal dynamics of plant growth responses to algal biomass? to better propose mechanisms by which plants acquire nutrients from algal biomass and thereby study and promote those processes in future agricultural applications. Data from various sources were transformed and used to reconstruct the nutrient release from the algae  The objective of this paper was to resolve the kinetics of how quickly nutrients including phosphate from algae biomass becomes available and is taken up by wheat plants compared to mineral nutrient sources. Identified patterns will serve as scheme aiming at using algae biomass as fertilizers in future agricultural applications.2. Environmental limitations such as growing temperature, pH, and culture densities depend on species. Algae are highly effective producers of organic material and do not compete with agricultural production for arable land. Multiple purposes for algae have been identified including producers of fuel, oil, gas or direct combustion, food and feed, pharmaceutical compounds, plastic, polymer compounds, and fertilizer. While most of these applications are feasible, the majority are not currently economically viable.Freshwater algae are ubiquitous and can be grown in media of varying quality, from sterile through to different types of wastewater. They require only water, light, nutrients, and COThe production of algae biomass as a fertilizer has dual value; the use of algae for the purification of wastewater coupled with the application of obtained biomass as fertilizer. The underlying concept is circular economy fertilization and has been reviewed before . VariousThe ability of algae to accumulate nutrients, directly and specifically, make them a valuable vector for recycling nutrients in agricultural systems. Algae can store excess phosphorus (P) and nitrogen (N) inside their cells resulting in high amounts of P and N that can be stored in vacuoles or the cytosol . Excess Phosphorus and N fertilization increase the risks for environmental pollution. However, they are the most essential macroelements for crop growth and therefore their acquisition and application are critical political and resource issues for global food and environment security . AlthougChlorella, were shown to improve growth and metabolic activity in willow  algae biomass is a fertilizer that will release nutrients over time, and (2) algae biomass may be a source of other compounds that plants respond to in terms of growth. The increasing research into the use of algae as a biofertilizer has not resolved the timing of release and growth responses. Resolution of kinetics of nutrient release from algae biomass and the subsequent uptake by plants is expected to help reveal which forms of P are directly or indirectly accessed by the plant, and thereby improve our prediction about what plant stage and time in cropping seasons responses can be expected by a farmer.Here we ask the question: what is the temporal dynamic of plant growth responses to algal biomass? To address this question, we combine the time resolved wheat leaf growth data from our group  with eleData for this paper were directly from Triticum aestivum L., var. Scirocco, KWS SAAT SE, Germany) growth responses to algae biomass  compared to mineral fertilizer sources in pots, in a glasshouse for 55 days after sowing (DAS) (Treatments in 2PO4 and (NH4)H2PO4 as mineral P , or algae [grown without nutrient limitations and harvested at 1 g (DW)∗L–1]. The algal biomass was either “dried” (spray-dried), or “wet” (with some living cells), and applied as high + 1x dried or wet algae biomass (115 or 130 mg P per plant), or low + 0.1x dried or wet algae biomass (11.5 or 13 mg per plant), for dried and wet algae respectively. All data from the 0.1x nutrient level treatments were not included in the publication of Briefly, 2) during the entire growth period . At harvest, 55 DAS, leaf area and shoot fresh weight were measured, dry weight of shoots and roots was recorded, and individuals, five per treatment, were analyzed for nutrient content.Ten wheat shoots per treatment were repeatedly imaged using an automated crane system  to compare to other published data . The root system size before the destructive harvest was estimated by correlating root dry weight and projected leave area  and R . The values for individual plants were either used directly or the arithmetic mean, and the corresponding error of the mean were calculated to represent the whole sample set. The growth function fitting to the data points was performed in Excel, using he means  in R to he means . They weR2 = 0.9925), averaging 2.65 mg P per g shoot dry weight, and accumulated up to 15 mg of shoot P. The second group, “high P content,” was grown in soil 2 + 1x mineral fertilizer, soil 2 + 1x dried algae biomass, or in soil 1 + 0 (positive control for high nutrient content), had a linear, but less pronounced correlation to shoot dry weight (R2 = 0.7958), averaging 5.56 mg P per g shoot dry weight, and accumulated up to >45 mg of shoot P.Two distinct groups of plants were observed at harvest (55 DAS) based on the relationship between shoot P content and dry weight in the experiment published in 2, at 30 DAS (linear R2 = 0.84) compared with 1238 cm2 in soil 1 at 55 DAS.The growth dynamics of wheat shoots over the whole experimental period  and lasted for 30 DAS. During phase 1, the growth trajectories were similar and differences between the nutrient sources were marginal. At 26 DAS growth trajectories started to diverge. The second phase was linear (R2 = 0.99–0.97) and occurred between 30 and 55 DAS. During phase 2, the growth rates (slopes) varied. For example, plants in soil 2 + 1x dried algae biomass had more leaf area at the beginning of phase 2 compared to those with soil 2 + 1x mineral fertilizer, but mineral-fertilized plants gained leaf area much quicker, reaching that of plants grown in soil 1 + 0. Plants with soil 2 + 1x wet algae biomass had the lowest leaf area in phase 2 but grew more quickly than plants grown in soil 2 + 1x dried algae biomass. Statistically significant differences were found between fitted growth functions of the negative control (soil 2 + 0) and all other nutrient sources in both growth phases (adj. p < 0.001), between soil 2 + 1x wet algae biomass and soil 2 + 1x dried algae biomass (adj. p ≤ 0.001) in the exponential growth phase. Soil 2 + 1x dried algae biomass and soil 2 + 1x mineral fertilizer (adj. p = 0.019), as well as soil 2 + 1x dried algae biomass and soil 1 + 0 (adj. p ≤ 0.001) differed in the linear phase, there were also differences between soil 2 + 1x wet algae biomass to soil 2 + 1x mineral fertilizer (adj. p = 0.044) and soil 2 + 1x wet algae biomass and soil 1 + 0 (adj. p ≤ 0.001), when comparing their growth trajectories  exhibited an increase in growth rate for all nutrient sources again. The exact time of the turning point between growth rates differed between nutrient sources. The plants grown in soil 1 + 0 showed a lower slope than the plants grown in soil 2 + 1x mineral fertilizer, soil 2 + 1x dried algae biomass and soil 2 + 1x wet algae biomass.The relative leaf area growth rate was calculated to further characterize the time-resolved image data from We calculated P uptake from different origins [, Phosphorus uptake and P contents over time varied among nutrient sources, at three obvious timepoints: beginning of the linear phase at 9 DAS, at the end of the linear phase at 30 DAS, and at the end of the exponential phase at 55 DAS . InitialWe aimed to answer the question: what is the temporal dynamic of plant growth responses to algal biomass as a sole P source? We conclude that for wheat, in a study that had temporally resolved phenotypic data, phosphorus from algal biomass was directly available and released gradually over time, leading to comparable growth to mineral fertilizer, albeit lower total P uptake.We used differences in leaf growth dynamics of wheat plants between dried or wet algal or mineral nutrient sources to map out the availability of P. The growth data indicated that, after the seed storage ran out, an initial amount of P was directly available in the early growth phase from all different nutrient sources and could support normal plant growth up until 55 DAS. Algae fertilizer, therefore, was unexpectedly found to be sufficient to support wheat development from the beginning of vegetative growth to beginning of heading, even in marginal soils such as the nutrient-poor soils 2 and 3 used here. We found, however, differences in growth dynamics between fertilized plants and those grown in soil 2 alone, and between dried and wet algae related to uptake dynamics between 30 and 55 DAS. These dynamics are discussed here.The positive relationship between P in the plant and its biomass that is reported here  is knownSimilarly, the high and low tissue P contents in The linearity of shoot P and shoot dry weight that we observed in the plants grown with low nutrient levels and wet algae, was not as strong for the plants grown with high nutritional levels . This maP values used for our calculations were literature-based and will need experimental verification in the future, because different varieties and growth conditions of mother plants influence the amount and quality of the seed storage in wheat , however, was maximally 0.11 m/cmOur results implicate that, in addition to seed P, an initial amount of P was available early on from all nutrient sources. We assume that residual algal growth media that might still have been attached after a rinse with tap water  was not Chlorella sp. (We developed a time and space resolved concept of P uptake  based onella sp.  and the ella sp. , becauseella sp. , as descella sp. .Chlorella vulgaris cell walls are especially robust, and the fast availability of their nutrients is therefore rather surprising (The mineral fertilizer, especially Hoagland solution, will contain free phosphates and it will, upon application, be available at once. Free phosphates on the other hand can easily become adsorbed, immobilized or leached out of reach, in a time dependent manner, and the longer it is in solution in high amounts the larger the portion that is unavailable for the plant . Living rprising . The frerprising  and simirprising , which irprising , unless rprising . The easThe model presented in The demonstrated fertilizing potential of algae depends on the release of P compounds readily available to wheat, and some of these forms are taken up from plant establishment. Additional studies of these different compounds and their temporal release will help to understand how we can utilize algae biomass as a fertilizer in the future. An important aspect will be to understand the plant mechanisms behind the P availability from algae for their future application as a fertilizer, along with the role of N in the fertilizing capacity of algal biomass. N volatility and the high requirements of wheat plants makes it another detrimental factor in environmental eutrophication.The original contributions presented in the study are included in the article/LM conducted all analyses, conceived figures, and wrote the manuscript. JK helped to writing the manuscript and the figures. RW helped to construct The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present datasets reveal that to assess the suitability of groundwater quality for drinking and irrigation uses in both Pre and Post Monsoon Season in Sarabanga River region, Tamilnadu, India based on various water quality indices. A total of 50 groundwater samples were collected in different location in a research area. Water Quality Index (WQI) is a number which indicates the suitability of water for drinking purpose. Sodium Absorption Ratio (SAR), Permeability Index (PI), Residual Sodium Carbonate (RSC), Percentage Sodium (%Na), Kelly Ratio (KR) and Magnesium Hazards (MH) are index value which elaborates the fitness of groundwater for agriculture uses. The WQI value for groundwater in both seasons reveals that 74.5 sq.km and 37.24 sq.km of the area were unfit for domestic purposes. Based on irrigation indices, almost all sample locations are suitable for irrigation purposes. The dataset demonstrates how water quality indices would be applied to policymakers to manage, handle and sustainably improve society at large. The dataset in this research paper reveals the hydrochemical properties of groundwater and its nature for drinking and irrigation purposes in the Sarabanga river region. A Sarabanga river flows through the Omalur taluk, Salem District in the state of Tamil Nadu, India . Omalur 2In order to assess the groundwater quality for drinking and irrigation purpose, a total of 50 groundwater samples were collected from a bore well at an average depth of 120 feet in river region during the pre-monsoon and post-monsoon seasons (the year of 2017). Samples were collected in a washed and dried polythene bottles at a capacity of 1000ml. Collected samples were kept at 4 °C and it transferred to the laboratory immediately for further analysis. The hydrochemical properties of groundwater were analyzed for the concentration of hydrogen ions (pH), total dissolved solids, alkalinity, Hardness, major cation like calcium magnesium, sodium, potassium and anion concentrations like chloride, sulphate, bicarbonate using Standard procedure APHA  were fol2.11.i was assigned to parameters that has a significant health effect [− was assigned the highest wi followed by SO42−, NO3−, Ca2+, Cl−, TDS, Mg2+, Na+, and K+ as shown in 3−. Each parameter has been assessed according to relevance in drinking quality of groundwater  is computed by the following equation The relative weights  assessed the suitability of groundwater for drinking purposes and compared the values of different water quality parameters with those of the World Health Organization  and the WQI range suggested by Ref.  was used


EDN1 gene is involved in AMS remains to be elucidated. A total of 356 healthy male soldiers who had not traveled to high altitudes in the previous 12 months were enrolled in our study. All participants were taken by plane from 500 m (Chengdu in Sichuan Province) to a 3700 m highland (Lhasa) within 2 hours. Clinical data were collected within 24 hours, and pulmonary function parameters were completed simultaneously. Genotypes were obtained by using iMLDR genotyping assays. A total of 237 soldiers (66.57%) presented AMS symptoms, including headache, dizziness, gastrointestinal upset and fatigue. Soldiers with AMS showed an increase in heart rate (HR), plasma tryptophan and serotonin, and a decrease in SaO2, FEV1, PEF, FVC, V75, V50, V25 and MMF . Notably, allele T in single nucleotide polymorphism (SNP) rs2070699 showed a positive correlation with the occurrence of AMS. A general linear regression analysis showed that rs2060799, Mean Arterial Pressure (MAP), SaO2, FVC, tryptophan and serotonin were independent predictors for the occurrence of AMS. Importantly, the area under the curve (AUC) values for tryptophan (0.998), serotonin (0.912) and FVC (0.86) had diagnostic specificity and sensitivity. Our results demonstrated that AMS is accompanied by changes in lung function parameters, increased plasma tryptophan and serotonin levels, and that the EDN1 polymorphism is a potential risk factor for AMS.Previous investigations have indicated that environmental and genetic factors collectively contribute to the development of acute mountain sickness (AMS), but whether the  AMS may further develop into high-altitude brain edema or high-altitude pulmonary edema, which endangers lives and reduces the working ability of populations in a highland environment; in addition, it can even cause obstacles to human migration4. Therefore, AMS has also become a public health problem to be solved.Acute mountain sickness (AMS) is the most common form of acute altitude illness and typically occurs in unacclimated persons ascending to altitudes >2500 m6. The most important environmental risk factors for the occurrence and development of AMS include the rate of elevation, the altitude at arrival, and the susceptibility of the individuals8. In addition, numerous studies have suggested that some people appear to be predisposed to developing altitude illness, suggesting a genetic contribution to susceptibility12. Genome-wide association studies and other genetic investigations have found that some genes  regulated through the hypoxia-inducible factor (HIF) pathway are closely related to hypoxia adaptation in native Tibetan and Andean populations at high altitudes12. More importantly, the EGLN1 gene polymorphism is associated with high-altitude adaptation and susceptibility to high altitude brain edema (HACE) in Aryan and Ayurveda populations14.Previous investigations have indicated that environmental and genetic factors collectively contribute to the development of AMS17. More importantly, research has indicated that the EPAS1 and EGLN1 proteins interact with endothelin-1 , and ET-1 variants have independent and interactive roles in the susceptibility to high altitude pulmonary edema18. ET-1 activation of Rho kinase (ROCK) decreases lung alveolar and vascular growth and contributes to the development of experimental bronchopulmonary dysplasia19. In addition, some studies suggest that ET-1 is an independent predictor of both AMS and its severity21. Therefore, the hypothesis is that the EDN1 gene interacts with the EPAS1 and EGLN1, which together contribute to the development of AMS. Current research aims to investigate the polymorphisms of the EDN1 gene associated with AMS in a large Han Chinese soldier population, which may contribute to the prediction of the development of AMS.Previous work demonstrated that polymorphisms of the EPAS1 and EGLN1 genes are associated with susceptibility to acute mountain sickness (AMS) in a Han Chinese populationA total of 356 healthy male soldiers  who had not traveled to high altitudes in the previous 12 months were enrolled in our study according to the inclusion and exclusion criteria. Subjects with any one of the following conditions were excluded: subjects took medication or received an intervention; subjects suffered from migraines, autoimmune diseases, respiratory diseases, or cardiovascular diseases; subjects had malignancy or liver and kidney dysfunction; subjects had an active infection or a bad cold; and subjects had psychiatric disorders or neuroses that interfered with the completion of the questionnaires performed by two trained doctors. 30 peoples as a unit were recruited and informed to taken to the test site simultaneously. All subjects accepted health education and instructions so that all of them could better understand the meaning of the experimentation. A series of data such as questionnaires, symptoms, auxiliary examinations, and blood were collected and scheduled from 9:00 to 12:00 on the same day.The participants’ baseline parameters  were measured at 500 m (Chengdu). All subjects ascended to the 3700 m highland (Lhasa) within 2 hours from 500 m (Chengdu in Sichuan Province) by plane. All participants completed the structured case report form (CRF) questionnaires, including self-reported demographic data, physiological symptoms, history of smoking and drinking and symptoms related to AMS according to the Lake Louise scoring system (LLss). Morning fasting venous blood was collected to isolate genomic DNA. Spirometric parameters were tested with a Sensor MedicsVmax229D pulmonary function instrument. Research parameters were recorded at 24 hours after the participants’ arrival at the 3700 m highland.www.chictr.org; No. ChiCTR-RCS-12002232.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Participants who agreed to participate in the study were familiar with the purpose and process of this study and signed informed consent forms before the trial. Informed consent was obtained from all individual participants involved in the study. All study protocols were approved by the Ethics Committee of The Second Affiliated Hospital of the Army Medical University and were carried out in accordance with established national and institutional ethical guidelines regarding the involvement of human subjects and the use of humans for research. Current clinical trials were registered in Chictr.org; URL: 23. The Lake Louise AMS questionnaire contained four different symptoms, headache, gastrointestinal symptoms, fatigue and dizziness, and each was graded from 0 to 3 in severity. The AMS consensus committee suggested no AMS as 0–2 points, mild AMS as 3–5 points, moderate AMS as 6–9 points, and severe AMS as 10–12 points23.For diagnosis, AMS was defined according to the 2018 Lake Louise AMS score as a total of three or more points from the four symptom categories, including at least one point from headache after a recent ascent or gain in altitudeAfter the participants arrived at 3700 m, four physiological parameters such as systolic blood pressure (BP), diastolic blood pressure (DP), serum oxygen saturation (SaO2) and heart rate (HR), were measured after resting for 30 min in a sitting position. The BP, DP and HR were measured by a sphygmomanometer . SaO2 was measured by a pulse oximeter . Mean artery pressure (MAP) was calculated by using the following formula: 25. As previously reported, the flow velocity index  for pulmonary function was corrected by air pressure using the following formula: 26.Pulmonary function parameters included forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and peak expiratory flow (PEF), forced expiratory flow at 25–75% of forced vital capacity , and maximum mid-expiratory flow (MMF) were measured with a portable spirometer . Each measurement was repeated twice, and the average value was used for statistical analysis. Spirometry was performed according to the guidelines of the American. Thoracic SocietyEDN1 gene were selected based on the SNP tagging of Han Chinese in Beijing from the HapMap database (http://hapmap.ncbi.nlm.nih.gov). The coverage of tagged SNPs, which was calculated by Tagger Server (http://www.broadinstitute.org/mpg/tagger/), averaged 70% of the information in the targeted region with a max r2 ≥ 0.8. Fasting venous blood (5 ml) was collected from each subject with EDTA-K2 anticoagulant, and the whole blood and plasma were separated and stored in a refrigerator at −80 °C. Genomic DNA was isolated from peripheral blood cells using a TIANamp Blood DNA kit  according to the manufacturer’s instructions. Quality control of the DNA was conducted by agarose gel electrophoresis and λ DNA-Hind III digested bands. The SNPs  in the promoter region of the EDN1 gene were genotyped in subjects, which was performed with improved multiple ligase detection reaction (iMLDR) genotyping assays with technical support from the Center for Genetic and Genomic Analysis . The levels of tryptophan and serotonin in the plasma were measured using a high-performance liquid chromatography (HPLC) system equipped with a Shimazu RF-10 AXL fluorometer detector set at excitation and emission wavelengths of 285 nm and 345 nm, respectively.A total of 3 single nucleotide polymorphisms (SNPs) within the promoter region of the http://www.R-project.org/). Principle component analysis (PCA) was done by using R software. The Hardy–Weinberg equilibrium was calculated using theonline software PLINK. The allele and genotype distributions of the 3 SNPs within the EDN1 gene between the AMS and non-AMS groups were calculated with PLINK (http://zzz.bwh.harvard.edu/plink/)27. A backward selection procedure in a stepwise regression analysis was employed to analyze the factors influencing the occurrence of AMS by using R software. Bonferroni correction in the present study minimized false positive associations to assure reliable results, although this method may have led to a relatively conservative result28.Clinical features are presented as the means ± standard deviations (SDs). A chi-squared test or Fisher’s exact test was used for the analysis of contingency tables depending on the sample size. Student’s t test and the Wilcoxon test were used to compare clinical characteristics between AMS and non-AMS groups by using R software  was used to determine the rationality of clinical characteristics of all participants from all participants. A total of 356 healthy male soldiers with an average age of 23.05 ± 3.99 were enrolled in the current investigation. Twenty-four clinical characteristics, including age, height, weight, and parameters of pulmonary function, were included in the current analysis. Pearson’s correlation was analyzed for these features, and the plot is presented in Fig. 2, FEV1, PEF, FVC, V75, V50, V25 and MMF in soldiers with AMS  had a positive correlation with the occurrence of AMS  within the EDN1 gene region were significantly different between soldiers with AMS and those without AMS . Genotypic and allelic frequencies of one SNP rs2070699) within 070699 wiFour clinical features  showed different distributions between SNP rs2070699 genotypes Fig. . rs207062 , FVC , tryptophan  and serotonin  remained independent predictors of AMS , MAP , SaO2, FEV1, PEF, FVC, V75, V50, V25 and MMF. More importantly, allele T in SNP rs2070699 denoted a positive effect on AMS, and general linear regression analysis revealed that rs2060799, MAP, SaO2, FVC, tryptophan and serotonin remained independent predictors of AMS. To our knowledge, this is the first study to investigate the factors influencing the occurrence of AMS in a Han Chinese population.In the current study, 356 healthy male soldiers endured a sharp ascent from 500 m to 3700 m within 2 hours. In this process, 237 soldiers (66.57%) endured AMS symptoms, including headache, dizziness, gastrointestinal upset and fatigue. Soldiers with AMS showed an increase in HR, tryptophan, and serotonin and a decrease in SaO28. In our primary reports, 66.57% of soldiers endured AMS after sharply ascending to a highland within 2 hours, which was a higher incidence than reported in other investigations. Therefore, investigating the potential factors that influence AMS is necessary.The standardized diagnostic clinical criteria for AMS were determined by the Lake Louise scoring system (LLss), which uses several symptoms for clinical diagnostic criteria. The Lake Louise score was developed at the International Hypoxia Conference in Lake Louise and is an internationally accepted standard for the diagnosis of AMS. AMS is characterized by nonspecific symptoms and distinct physiological outcomes. The main symptoms of AMS are headache, anorexia, nausea, vomiting, fatigue and dizziness, but not all will be present at the same time. Headache is an essential symptom for the diagnosis of AMS. The symptoms of AMS generally occur 6 to 12 hours after arrival at high altitudes. The LLss mainly employs four symptoms for scoring including headache, dizziness, gastrointestinal reactions and fatigue. After reaching high altitude, those with a score greater than or equal to 3 points and an accompanying headache received a diagnosis of AMS. If patients had a score of greater than or equal to 3 points and less than 5 points, they were diagnosed with mild AMS. Similarly, a score of greater than or equal to 5 points was recognized as severe AMS. According to the criteria, a previous investigation indicated that approximately 10% of individuals will suffer from AMS at 2500 m, and the incidence of AMS will exceed 60% at 4500 m30. Previous studies have shown that acute highland disease generally occurs after a person rises to 2500 meters above sea level, and its incidence and severity increase with altitude. At an altitude of 2500 meters, the incidence of acute high-altitude disease is approximately 10% to 25%, and symptoms are usually mild. However, at 4500 to 5500 m, the incidence of acute high-altitude sickness is between 50% and 85%31. A retrospective study showed that the main independent risk factors for acute high-altitude disease include a history of acute high-altitude disease, a rapid rise , and a lack of previous exercise32. Other possible risk factors include female sex, age under 46 years, and a history of migraines. Exercise may aggravate acute high-altitude disease, but good physical fitness is not a protective factor. A meta-analysis by Vinnikov D showed that smoking may also be a risk factor33, but there was no significant difference in body mass index, age, or smoking and drinking status between the AMS group and the non-AMS group after high-altitude exposure, indicating that our sample homogeneity was relatively good. However, our current report indicated that the rs2070699 polymorphism within the EDN1 gene was an independent predictor of AMS, which was notable.The range of elevation is the main factor for AMSEDN1 gene is a possible susceptibility factor that influences AMS. The frequency of the T allele of rs2070699 in the EDN1 gene is 0.545 from the 1000 genome database. Data from current research indicated that the T allele frequency was 0.5365 (191/365), which denotes no differential distribution. However, in the AMS group, the T allele frequency was 0.571 (271/474) and was highly enriched in the AMS group . The mechanism behind EDN1 and the occurrence of AMS still needs further investigation. Previous studies may offer some clues about the connection between EDN1 and AMS. Serotonin is an important physiologically active substance in the body. The precursor of serotonin is tryptophan. Tryptophan becomes serotonin under the catalysis of enzymes in the body. Serotonin has a strong contractile vasoconstriction in the lungs as well as in peripheral blood vessels. In addition, serotonin can increase the vasoconstrictor effects of other vasoconstrictors 35. Determination of serum tryptophan and serotonin concentration can indirectly reflect the body’s vasoconstriction. Research suggests ET-1-ROCK interactions have already been proven to contribute to decreased alveolar and vascular growth and pulmonary hypertension (PH) in experimental bronchopulmonary dysplasia36 confirmed that ET-1-mediated positive inotropic effects and myocardial fetal gene induction further lead to PH. Interestingly, recent reports from Josefa et al. demonstrated that melatonin, which is an indoleamine that is synthesized from tryptophan under the control of the enzymes aryl alkylamine N-acetyltransferase (AANAT) and acetyl serotonin methyltransferase (ASMT), can reduce endothelin-1 expression through the inactivation of FoxO-1 and NF-κβ, playing an important role in colon cancer37. Coincidently, our results show that tryptophan and its metabolite product serotonin are more highly expressed in the AMS group than in the non-AMS group. Therefore, tryptophan and its metabolism may influence the expression of EDN1 through the inactivation of FoxO-1 and NF-κβ expression, which further leads to AMS. However, whether tryptophan and serotonin regulate the expression of EDN1 requires further validation.Current reports have demonstrated that rs2070699 of the EDN1 gene were considered in a relatively small sample, which may influence the effect size in the association study. Second, the current study did not perform a functional experiment for the rs2070699 genotype in the expression of the EDN1 protein; therefore, direct evidence to show that rs2070699 regulate the expression of EDN1 is lacking.Two limitations in the current report must be mentioned. First, only select SNPs of the In summary, our results demonstrated that AMS is accompanied by changes in lung function parameters, increased plasma tryptophan and serotonin levels, and that the EDN1 polymorphism is a potential risk factor for AMS.


Poor dietary choices are major risk factors for obesity and non-communicable diseases, which places an increasing burden on healthcare systems worldwide. To monitor the effectiveness of healthy eating guidelines and strategies, there is a need for objective measures of dietary intake in community settings. Metabolites derived from specific foods present in urine samples can provide objective biomarkers of food intake (BFIs). Whilst the majority of biomarker discovery/validation studies have investigated potential biomarkers for single foods only, this study considered the whole diet by using menus that delivered a wide range of foods in meals that emulated conventional UK eating patterns. Fifty-one healthy participants  followed a uniquely designed, randomized controlled dietary intervention, and provided spot urine samples suitable for discovery of BFIs within a real-world context. Free-living participants prepared and consumed all foods and drinks in their own homes and were asked to follow the protocols for meal consumption and home urine sample collection. This study also assessed the robustness, and impact on data quality, of a minimally invasive urine collection protocol. Overall the study design was well-accepted by participants and concluded successfully without any drop outs. Compliance for urine collection, adherence to menu plans, and observance of recommended meal timings, was shown to be very high. Metabolome analysis using mass spectrometry coupled with data mining demonstrated that the study protocol was well-suited for BFI discovery and validation. Novel, putative biomarkers for an extended range of foods were identified including legumes, curry, strongly-heated products, and artificially sweetened, low calorie beverages. In conclusion, aspects of this study design would help to overcome several current challenges in the development of BFI technology. One specific attribute was the examination of BFI generalizability across related food groups and across different preparations and cooking methods of foods. Furthermore, the collection of urine samples at multiple time points helped to determine which spot sample was optimal for identification and validation of BFIs in free-living individuals. A further valuable design feature centered on the comprehensiveness of the menu design which allowed the testing of biomarker specificity within a biobank of urine samples. The amount and pattern of foods and beverages consumed influence gene expression  and are www.foodb.ca) and of selected food components, e.g., Phenol Explorer (developed by INRA: www.phenol-explorer.eu). However, when foods are consumed, they undergo metabolic transformation during digestion and absorption by enterocytes and colonocytes, by bacteria within the gut lumen and by Phase 1 and Phase 2 enzymes within the liver. Therefore, although the patterns of metabolites present in blood or urine reflect what has been consumed, the specific metabolites in such body fluids may differ substantially from those ingested (Biomarkers of food intake (BFIs) assessed in body fluids or easily-accessible tissues offer potential alternative, objective routes to estimating dietary exposure . Measureingested .We, and others, have applied metabolomics approaches to blood, saliva and urine in human studies to discover novel BFIs , 15–22. The MAIN  Study was designed to address these challenges by investigating biomarkers of food intake under conditions in which study participants consumed well-characterized foods within conventional diets with respect to meal design, cooking and eating patterns and collected urine samples at home without changing their usual behavior. This provided an opportunity to expand biomarker coverage to include a more comprehensive range of foods and beverages that are highly consumed in the UK and considered important for the UK government healthy eating policy . Our priThe studies involving human participants were approved by East Midlands—Nottingham 1 National Research Ethics Committee (14/EM/0040) following Proportionate Review. Caldicott approval for storage of data and data protection was granted by Newcastle-upon-Tyne Hospitals NHS Foundation Trust [6896(3109)]. The trial was adopted into the UK Clinical Research Network (CRN) Portfolio (16037) and was registered with International Standard Randomized Controlled Trial Number (ISRCTN), 88921234.A study information sheet was given to all potential participants in advance of their first visit to the research unit. The participants provided written informed consent to participate in each study, taken by an appropriately trained researcher. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Participants were recruited using the inclusion criteria in a priori exclusion list , wine (fermented), grape juice (pasteurized/heat-treated/concentrated), sparkling grape juice (carbonated drink), and a fruit smoothie (complex beverage). Whole grains were delivered across both experimental periods in the form of rye bread , wholemeal bread (toasted and untoasted), wholegrain breakfast cereal (Weetabix®), porridge oats (boiled/microwaved with milk) and wholemeal pasta (extruded). Participants consumed a range of commonly eaten foods in three different daily menu plans, which were presented in a different order in each of three experimental weeks. This design facilitated the search for novel biomarkers of specific foods, but also provided the potential to characterize the kinetics of biomarker appearance and decay and to investigate the longevity of biomarker signals in urine.Each daily menu plan was designed to emulate conventional UK eating patterns with a breakfast, lunch, afternoon snack and dinner. All foods and drinks for the whole intervention were provided to participants in appropriate portion sizes and with cooking instructions, where necessary. Participants were encouraged to consume these items to the exclusion of any other foods or beverages, but had the freedom to eat or not the meals and to interpret the cooking instructions as they wished. During each experimental week, the allocated menu plans were followed from Tuesday to Thursday, as shown in During the “Pre” day, participants were asked to restrict, as far as possible, their polyphenol intake. In practical terms, this meant reducing their intake of brightly colored fruits and vegetables, chocolate, tea, herbal teas and coffee. Participants were asked to abstain from alcohol and stop taking any dietary supplements for the duration of the studies. In Study 1, dinner on the “Pre” day was either a standardized evening meal or a low polyphenol meal of the participant's choice whilst in Study 2 it consisted of one of three meals designed to capture biomarkers of relatively unhealthy food choices, particularly poorer-quality meats and breaded and battered foods.Menu plans were developed using Public Health England policy advice from The Eatwell Plate which has been revised as The Eatwell Guide  and infoTo identify the easiest to collect and most informative spot urine sample, participants were asked to collect several spot urine samples each day. These included their first morning void (FMV) before breakfast; a fasting sample, defined as a sample collected after the FMV but before breakfast, following an overnight fast of at least 12 h; a sample collected any time between finishing breakfast and eating lunch and a sample collected any time between finishing lunch and eating their afternoon snack. Each urination between dinner and the FMV the following day was also collected. To reduce variation in timing of urine sample collection, participants were encouraged to consume meals within a 2 h time slot  or 30 min time slot (afternoon snack) as illustrated in ad libitum. To help maintain good hydration, participants were given eight 500 ml bottles of water per experimental week, one to be drunk with/after each dinner, one to be drunk during each experimental day and one to be drunk on the morning of the “Post” day, prior to venipuncture.Participants were encouraged to keep hydrated and to drink water Participants in Study 1 were randomized (random.org) at enrolment to one of two dinner options on the “Pre” day. In Study 2, participants were randomized to one of 12 3 × 3 Latin squares  at enrolment. The order in which these participants consumed the three less healthy dinners on the “Pre” day in each of the three experimental weeks was randomized independently.Dietary compliance was assessed by participant self-report. They were asked to record how much of each food/drink item they ate. If some food was uneaten, the participant recorded the amount eaten as 75, 50, 25% or 0, as appropriate. Dietary substitutions, intrusions and protocol deviations were also recorded. Participants recorded if they substituted any foods or drinks of their own choosing for those provided by the research team, ate or drank any additional items or prepared any of the meals differently from that instructed.A 1-day food diary was used to record all foods and drinks consumed from 8pm on the evening before and throughout the “Pre” day. This was used to check participant compliance with the low polyphenol diet prior to commencement of the experimental period each week.To monitor compliance with both the urine collection protocol and suggested meal times, participants completed a Urine sample collection record and a Meal time record—the latter asked specifically at what time they finished eating each meal. This acted as a check on the accuracy of the FMV and fasting urine samples collected. In addition, this information allowed calculation of the time interval between meal consumption and subsequent urine collections.Information on participant socio-demographics , medical history, current medications, and use of dietary supplements was collected at enrolment.In Study 1, weight, height and waist circumference were measured on “Pre” and “Post” days of each experimental week. In Study 2, height and waist circumference were recorded in week 1 only, whilst weight was recorded weekly, pre- and post- the experimental period. All anthropometric measurements were made at the research unit by the same researcher.Body weight was measured to the nearest 0.1 kg using a Tanita body composition analyzer ; height was measured to the nearest 0.1 cm using a Leicester portable height measure . Waist circumference  was measured to the nearest 0.1 cm using a non-stretch tape measure over bare skin, whenever possible. Waist circumference measurements were taken in duplicate, or repeated until two measurements agreed within 1 cm. Participants were asked to wear lightweight clothing and removed their shoes for all measurements.Habitual dietary intake was assessed at the beginning of each study using a locally adapted version of the validated food frequency questionnaire (FFQ) used in the European Prospective Investigation into Cancer and Nutrition (EPIC) .To monitor their physical activity (PA), each week participants were asked to complete the International Physical Activity Questionnaire (IPAQ) short form .Participants collected urine samples at suggested times  in a calWhole blood, serum and plasma were archived at −80°C for future lipidomic analysis and associated metabolite fingerprinting. Venipuncture was performed on the “Post” day of each week after a minimum 12 h overnight fast. After filling, blood tubes were inverted several times, kept flat on ice and processed within 2 h. Before processing, the height of the blood in each tube was recorded to assess filling. Plasma was separated from anti-coagulated blood collected in 1 × EDTA and 2 × lithium/heparin Vacutainer® blood collection tubes  by centrifugation at 3,100 × g for 5 min at 4°C in a Jouan CR3i centrifuge . Plasma was stored at −80°C in 1 ml aliquots in plasticiser-free microtubes as described above. Serum was separated from coagulated blood collected in 2 × gel Vacutainer® tubes at least 30 min after collection, aliquoted and stored as for plasma. One tube of unprocessed whole blood, collected in an EDTA-coated tube, was also stored at −80°C.A summary of the time points at which key data, measurements and biological samples were collected is given in t-test was employed for feature selection  allowed further structural identification of putative biomarkers as previously described  coupled to a Dionex Ultimate 3000 Ultra High Performance Liquid Chromatography (UHPLC) system (Thermo Scientific). Chromatographic separation was performed on a reverse phase (RP) Hypersil Gold 1.9 μm, 2.1 × 150 mm column (Thermo Scientific) using 0.1% formic acid in H2O (mobile phase A) and 0.1% formic acid in MeOH (mobile phase B) at a flow rate of 0.6 ml/min and column oven temperature at 60°C. Each sample (5 μl) was analysed by following a gradient after 0.5 min isocratic A to 40 % B in 3.5 min and subsequently to 100% in 5 min. The column was washed with 100% B for 2.5 min and re-equilibrated for 2.5 min. Data were acquired in two runs using respective positive and negative ESI mode. Each experiment consisted of a full scan  within a 1 s cycle time using selected targeted mass properties for either positive or negative ionization mode between 1 and 12 min runtime. The maximum injection time was 22 ms and the Automatic Gain Control (AGC) target of 1 × 104 was set to be exceeded if there is parallelisable time. The spray voltage was 3.5 kV for positive and 2.5 kV for negative ionization modes. The temperatures of the ion transfer capillary and vaporiser were, respectively, 342° and 258°C with sheath and auxiliary gas set at 45 and 13 arbitrary units, respectively. The data were acquired using Thermo Scientific Xcalibur version 4.2.28.14.The methodology used for biomarker identification has been described in detail elsewhere , 27. Forion tool . Ultra Hescribed . Refractn and retention times with authentic standards or with the respective aglycone (if the biotransformation product was unavailable). MSI level 2 structural assignment was achieved by putatively matching signal behavior with that of authentic standards reported in the literature  or fragmentation pattern alignment with data in public/commercial spectral libraries . MSIn = 105). More than 70% of those invited to participate in Study 1 had taken part in earlier nutrition research studies at Newcastle University and six were subsequently recruited. A further nine participants learned about the study through word of mouth. The majority (85%) of those who expressed an interest in Study 2 and were screened for eligibility (69%) had learned about it through local community and internal university advertising and contributed 66% of enrolled participants. The remaining participants had either taken part in Study 1 (17%) or heard about the study through word of mouth (17%). The desired sample size (n = 15) was met in Study 1 and the target recruitment (n = 30) was exceeded by 20% in Study 2.As shown in Participants aged 19–77 years mean 46 years), of whom 57% were female, took part in the studies . The par years, oParticipant characteristics were similar in both studies but those recruited to Study 2 were slightly older (mean 1.4 years) and had a lower BMI. They were less likely to report consuming alcohol, although weekly consumption amongst those who did was higher . Ten percent more participants were categorized as having central obesity in Study 1 than in Study 2.In recent publications, we have reported that dietary exposure biomarker discovery was possible within the context of the present comprehensive food intervention mimicking a typical UK diet in free-living individuals with minimal intrusion on normal daily activities , 27. Valm/z 204.98143. Detailed structural analysis indicated that this particular compound is pyrogallol sulpate , suggests that this food intervention strategy was highly acceptable to participants . This f2) and, again, it is unlikely that this infringement of the exclusion criteria will have affected the findings. It is possible also that participant genotype will alter the pattern of metabolites produced from a given food constituent and so for this reason further work should investigate the impact of common variants in the genes encoding Phase 1 and Phase 2 enzyme systems, an example being the P450 consortium —contrary to the study inclusion criteria—but it seems unlikely that this would have had a major effect on the overall findings. Similarly, our cohort included three participants who were slightly obese . Caldicott approval for storage of data and data protection was granted by Newcastle-upon-Tyne Hospitals NHS Foundation Trust [6896(3109)]. The trial was adopted into the UK Clinical Research Network (CRN) Portfolio (16037) and was registered with International Standard Randomized Controlled Trials Number (ISRCTN), 88921234. A study information sheet was given to all potential participants in advance of their first visit to the research center. Written informed consent was obtained prior to participation from each eligible individual, for each study, by an appropriately trained researcher. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Trial registration: ISRCTN, ISRCTN88921234. Registered 3rd April 2014—retrospectively registered, JD, JM, and MB conceived the study. JM, JD, NW, AL, and MB designed the study and menu plans. NW and LX undertook participant recruitment, developed participant handling protocols, ran the intervention study, supervised support staff, collected all biological samples, and refined sampling methodology. IG-P and EC coordinated project input from Imperial College London. NW and MS analysed and interpreted the data with respect to study design, participant recruitment and characteristics and participant and sample handling, and produced the tables and figures. KT and MB analysed the urine samples. AL analysed and interpreted the metabolomics data and produced the tables and figures. NW, AL, JD, and JM wrote the manuscript. JM and JD coordinated the project and supervised the research teams in Newcastle and Aberystwyth Universities, respectively. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


All composites were fabricated by pressure infiltration under the same conditions . For each matrix composition, two main factors due to the presence of the foam determined the composite microstructure—the efficiency of foam penetration and different conditions of metal crystallization. The lowest porosity was obtained when Cof45ppi was used and was independent of the applied matrix composition. The metallic component microhardness increased with a decrease in the carbon cell size as well as a decrease in the α-Mg grain size; both of those results should be taken into account during theoretical calculations. Compression and three-point bending strength measurements showed increases as the carbon cell size decreased, but reinforcing effectiveness relative to the matrix material depended on the metal matrix composition. At the fractured surface, different structural effects in the foam and matrix as well as at the interface were observed and depended on the foam geometry, metal composition and mechanical test type. In glassy carbon foam, those effects occurred as cracking across walls, fragmentation, and delamination, while in the matrix, shear bands and intergranular cracking were observed. On the delaminated foam surface, the microareas of a thin oxide layer were detected as well as dispersed phases characteristic for the applied matrix alloys. The accumulation of intermetallic phases was also observed on the metal matrix surface in microareas delaminated from the carbon foams. Mechanical property results indicated that among the tested, open-celled, carbon foams a 45 ppi porosity was the most useful for pressure infiltration and independent of magnesium-based matrix composition.Magnesium-based composites reinforced with open-celled carbon foams (C Ceramic–metal interpenetrating network materials are a group of composite materials that have previously defined component distribution and a defined microstructure that can be obtained by different technologies. Manufacturing solutions for the sintering of ordered ceramic–metal powder mixtures include conventional ,2 or SHSof) selection for a new magnesium matrix composite is described. The literature studies have shown that an application of open-celled ceramic materials of different porosities and cell sizes are proposed for metal matrix composites to improve stiffness, compressive strength and wear resistance [2O3 foam of porosity 90% and mean pore size 450 μm [3N4/Al–Mg system was reported [In this work, the problem of open-celled carbon foam  as well as ensuring enough high mechanical properties are strongly limited by manufacturing techniques. The second crucial technological challenge is components consolidation. As mentioned, pressureless, vacuum and pressure infiltrations can be used in composite manufacturing. Effective and nondestructive movement of the liquid metal in porous ceramics has influenced the chosen infiltration conditions  which determines the final composite porosity as well as the bonding character. The nature of that process is quite different from the fabrication of metal–ceramic composite foams, where the contact between liquid metal and the ceramic surface forms an intense metal mixture after which pore formation of metal-based suspension by inert gases occurs ,29,30,31of–Mg composite technology [of volume fraction (3%) in these composites, it was lower than conventional composites with microsized reinforcement as carbon fibers (23–70%) [In this work, a magnesium matrix composite with open-celled carbon foam obtained by pressure infiltration is reported; the role of cell size  for similar foams with an open porosity of ~97 vol %, in microstructure formation and improvement of properties was analyzed. The additional pressure was needed for system consolidation because the wettability of carbon materials by magnesium was unsuitable for spontaneous infiltration as discussed in a previous work from our group focused on Cchnology ,33. The chnology ,35 must (23–70%) ,37,38,39(23–70%)  and only(23–70%) ,42.The influence of cell size  was analyzed for pure magnesium and two commercial cast magnesium alloys AZ31 and RZ5. The more beneficial foam for the magnesium-based composite was indicated after microstructure characterization and mechanical properties measurements. In addition, the crystallization processes induced in the metal matrix by the presence of carbon cells is examined and discussed.of was called reticulated vitreous carbon  with a similar porosity 97%, but cells smaller than 100 ppi and a microstructure as presented in For composite fabrication, the carbon foams obtained at the Silesian University of Technology , Faculty of Materials Science had porosities of ~97% and cells of 20 and 45 ppi were applied as well as a 10 ppi to compare magnesium crystallization effects. As shown in As a metal matrix, technical grade elemental magnesium was chosen which avoided the influence of alloying elements, both in the analysis of the matrix surrounded with continuous solid carbon network crystallization and the processes at the solid–liquid interface. Additionally, two commercial magnesium alloys AZ31 and RZ5  were appFor components consolidation, independently on applied foam and matrix material, a Degussa press  was utilized and took place under vacuum (2.7 Pa). Components were heated initially in a graphite crucible up to 690 °C and liquid metal under a maximum pressure of 5 MPa was intruded into carbon foams. After 10 min, cooling  under pressure supported with water was applied. Composite samples with 30 mm-diameters 5 mm-thick were obtained.of–Mg composites with different-sized foams, where the magnesium grain size was characterized, samples were additionally etched with a solution containing: 33 mL H2O, 66 mL C2H5OH, 1 mL HNO3, 2 g citric acid, 0.1 picric acid to expose the grain boundaries. For each sample, 15 LM images were registered and measurements were conducted using the Met-Ilo program  after automatic and manual binary image correction. For AZ31 and RZ5 matrices, that procedure was intentionally skipped due to problems with the correct detection of α-Mg grains with procedures of the automatic image analyze. They were caused by the similarities in the shades of gray for magnesium grain boundaries and other phases seen in magnesium alloys.For determining the open porosity of fabricated materials, the Archimedes method was applied. Their microstructures were characterized by a light microscope , scanning electron microscope  and Hitachi S-3400 N, Thermo Electron Corporation, Waltham, MA, USA) with EDS . Polished cross-sectioned unetched samples as well as fractured samples were examined. For C0.2, Duramin-5 microhardness tester, Struers, Copenhagen, Denmark) were determined.For all composites, after 20 measurements, the matrix microhardness levels . For compressive strength measurements, based on the ASTM E9 standard , 5.0 × 5The fractured surfaces were examined with SEM to analyze the decohesion processes of composites and understand the differences associated with applying foams of the same porosity, but different cell sizes, as well as the matrix composition. Observations were focused on the phenomena occurring in foams, matrices, and interface.17Al12 phase accumulation, oxide phases and Al4C3 carbide formation. In the case of the RZ5 matrix, except for the magnesium oxide layer enriched with RE (rare earth elements) (lements) f–h the alements) i was obslements) g,h.of45ppi had the lowest porosity regardless of the applied matrix composition, but for the same cell size, it was the lowest for a matrix of pure magnesium. The strong porosity increase of (~9%) was characteristic for the Cof100ppi–RZ5 system and could have been caused by less effective infiltration induced by interphase phenomena, including carbon component destruction by magnesium alloying elements that were more intense for foams with more extended surfaces. That interaction problem has been previously reported for glassy carbon particles—RZ5 composite [Open porosity measurement results  demonstromposite .of–RZ5 composite, where the open porosity was also clearly higher. To explain the microhardness increase, the magnesium grain sizes in Cof–Mg composites (for completeness with data for Cof10ppi–Mg composite) were determined  initial cellular distribution, many cracks across the foam walls were detected, but the effect of glassy carbon refinement was less intense. At the fractured metallic surface, the multidirectional shear bands were visible as well as dendrites in some microareas  with three different cell sizes, pure magnesium, AZ31 and RZ5 as matrices were used, led to the following conclusions:of-Mg composites as materials in automotive and airplane constructions.The further examinations of the novel composites will be focused on microstructure and properties design, especially of fracture toughness, ability to energy absorption, thermal and electrical properties characterization. The results will show the suspected usefulness of C