Eupatorium buniifolium against poliovirus type 1 was determined by in vitro assays with an effective concentration 50 (EC50) of 23.3 ± 3.3 µg/mL. Bioassay-guided fractionation of the OE allowed the isolation of an active principle that was identified by spectroscopic methods  as the benzofuran euparin. The plaque reduction assay in Vero cells was used to assess the antiviral activity of euparin against poliovirus types 1, 2, and 3 with EC50 values of 0.47, 0.12, and 0.15 µg/mL, respectively. Moreover, this compound showed high selectivity indexes of 284.9, 1068, and 854.7, respectively. In order to identify the mechanism by which euparin exerts its antiviral activity, the virucidal effect, the pretreatment of Vero cells, and the time of action on one viral replication cycle were evaluated. Results obtained demonstrated that euparin exerts its effect during the early events of the replication cycle, from the virus adsorption to cells up to the first twenty minutes after infection. This is the first report on the presence of euparin in E. buniifolium and its antiviral activity.The antiviral activity of the organic extract (OE) of  Poliomyelitis is caused by poliovirus (PV), which can affect the nervous system causing permanent paralysis. PV is an RNA virus that belongs to the genus Enterovirus of the large family of Picornaviridae. There are three serotypes of PV . Two trivalent polio vaccines have been available since 1961: the intramuscular inactivated vaccine of Salk and the oral attenuated vaccine of Sabin. Both of them stimulate the production of neutralizing antibodies antipolioviruses that protect from the disease. Polioviruses have been eradicated from the United States in 1980 and from the Western Hemisphere in 1991. In 1999, the Global Polio Eradication Initiative wiped out PV-2. Nowadays, and according to the World Health Organization, poliomyelitis remains endemic in Afghanistan, Nigeria, and Pakistan and transmission has been reestablished in three countries which were previously declared as polio-free  ∗100.Antiviral activities of 3 cells/well; 24 h) in 96-well plates were exposed to twofold dilutions of OE, fractions F2–F4, and euparin in DMSO for 72 h at 37°C. Cellular controls were incubated under the same conditions in presence of growth medium without drug. Cell viability was calculated as [Abstreated  cells/Abscellular  control] × 100. The cytotoxic concentration 50 (CC50) is defined as the concentration that reduces cell viability by 50% with respect to cell control and is determined from dose-response curves. The maximum noncytotoxic concentration (MNCC) is the maximum concentration of OE, fraction, or euparin that leaves 100% of viable cells.Cell viability was assessed by the MTS/PMS test as previously described . Briefly2; 37°C) were infected with 100 PFU of PV-1/well in presence of serial twofold dilutions from the MNCC of E. buniifolium OE, fractions F1–F5, or euparin. Following 45 min of adsorption at 37°C, the viral inoculum was removed; cell monolayers were washed twice and overlaid with PM supplemented with the same concentrations of OE, fractions F1–F5 or euparin added during the adsorption period. Mock-infected cells and virus control were included. After 24–48 h of incubation cell monolayers were fixed and stained with 0.75% crystal violet in methanol : water (40 : 60) and viral plaques were counted. The effective concentration 50 (EC50) value is the concentration of OE, fraction, or euparin that reduces the number of viral plaques by 50% with respect to the viral control and was calculated by regression analysis of the dose-response curves generated with the data. Reduction of plaques (%) was calculated as [1 − (no.  plaques  treated/no. plaquescontrol  virus)] × 100. The selectivity index (SI) was calculated as CC50/EC50. The antiviral activity of E. buniifolium OE and euparin was also determined against PV-2, PV-3, HSV-1, HSV-2, and VSV by reduction viral plaque assays in Vero cells monolayers as described earlier.Confluent Vero cell monolayers seeded in 24-well plates  was incubated at room temperature (r.t.) or 37°C for 30 min in IM with or without 10 µg/mL (10X EC90) of euparin. The residual infectious virus particles were determined by viral plaque assays in Vero cell monolayers.PV-1 , cells were washed twice with phosphate buffer saline (PBS) and treated with 1.0 and 10 µg/mL of euparin in IM, corresponding to EC90 and 10X EC90, respectively. After 7 h incubation at 37°C, the medium was removed; cell monolayers were washed three times with PBS and infected with 100 PFU/well of PV-1 in 0.1 mL of IM. Following 45 min of adsorption at 37°C, the virus inoculum was removed and PM was added. After 24 h, cells were fixed and stained and viral plaques were counted.Pretreatment of Vero cells with euparin was evaluated as previously described . Vero ceµg/mL (1X EC90) were evaluated: (i) only during adsorption period (Adsorption); (ii) after adsorption and until the end of the experiment (After Adsorption), and (iii) during and after the adsorption (Throughout). Briefly, Vero cell monolayers cultured in 24-well plates were precooled for 1 h at 4°C. Cells were then infected with 100 PFU of PV-1 virus/well in presence or absence of euparin and further incubated at 4°C for 1 h. Cell monolayers were washed with PBS, and then PM with or without euparin was added. After 24 h, the viral plaques were counted.To study the effect of euparin in the adsorption and post adsorption events of PV-1 in Vero cells, three different treatments at 1.0 µg/mL of euparin was added at different hours after infection (h p.i.): 0, 1, 2, 3, 4, 5, 6, and 7 or at different minutes p.i.: 0, 10, 20, 30, 40, 50, and 60. Cells were further incubated up to 8 h. At this time, the supernatants were collected, clarified by centrifugation , and the extracellular virus production was determined by viral plaque assays. Confluent Vero cell monolayers cultured in a 24-well plate were infected with PV-1 (m.o.i. = 5) for 1 h at 4°C. Following the adsorption period, cells were washed three times, and 10 50 and CC50 values were calculated using GraphPad Prism programme v. 5.01. Statistical differences between different treatments were determined using one way ANOVA with Tukey's posttest. Differences were considered significant when the P value was less than 0.05. Data are presented as means ± SD of three independent assays. The ECE. buniifolium OE and AE was determined against PV-1 by the viral cytopathic effect reduction assay. The results obtained showed that only OE inhibited PV-1 replication at the two concentrations tested (25 and 100 µg/mL) with CPE reduction values higher than 50% (data not shown).The antiviral activity of 1–F5) which were assayed for their in vitro anti-PV-1 activity by the plaque reduction assay  was isolated and identified as euparin ; m.p. 120–122°C) by comparison of its spectral data with the literature values λmax (nm) MeOH: 260, 356; IR (KBr), νmax (cm−1): 3388 (OH), 1470 (aromatic), 1636 (C=COR); 1H NMR (MeOH-d4), δ (ppm): 8.18 ; 6.95 ; 6.78 ; 5.74 ; 5.21 ; 2.72 ; 2.14 . 13C NMR (DMSO-d6), δ (ppm): 204.60 , 161.24 , 159.49 , 157.64 , 132.45 , 124.27 , 121.91 , 116.79 , 112.23 , 102.54 , 98.24 , 25.59 , 17.85 ; EI-MS  218 (19.30), 217 (48.18), 216 (68.16) [M]+, 203 (2.52), 202 (16.22), 201 (100.00), 173 (26.88). Euparin presented the following spectral data: UV E. bunnifolium OE, fraction F2 and euparin, on Vero cells, was evaluated by the MTS/PMS assay and expressed as cell viability percentage. When cells were treated with OE, fraction F2, or euparin, CC50 values were 114.8 ±1.8 µg/mL, 254.2 ± 1.2 µg/mL, and 128.2 ± 2.2 µg/mL, respectively (The cytotoxic effect of ectively .E. buniifolium organic extract (OE), fractions F1–F5, and euparin against PV-1 was evaluated by the plaque reduction assay. As showed in 50 values for OE, fraction F2, and euparin were 23.3 ± 3.3 μg/mL; 13.3 ± 2.6 μg/mL and 0.47 ± 0.05 μg/mL, respectively. The SI values for OE, F2, and euparin were 5.5, 19 and 284.9, respectively. The antiviral activity of 50 values of 25.0 ± 1.8 μg/mL and 21.1 ± 2.5 μg/mL, respectively. Euparin showed EC50 values of 0.12 ± 0.01 µg/mL (SI = 1068) and 0.15 ± 0.01 µg/mL (SI = 854.7) against PV-2 and PV-3 respectively . These results suggested that euparin did not exert a virucidal activity against PV-1  with concentrations of euparin as high as 10 nst PV-1 .µg/mL of euparin to Vero cells monolayers 7 h before infection with PV-1 did not exert any reduction in viral infection. Thus, it can be deduced that no antiviral activity was induced in these cells during the pretreatment with euparin  of this compound during the different infection periods. The maximum inhibition was obtained when the compound was present in the postadsorption period or throughout the infection time  exerted antiviral activity (EC50 = 23.3 ± 3.3 μg/mL). In this context, we have evaluated the antiviral activity of the medicinal plant 1–F5), F2 showed the highest antiviral activity (EC50 = 13.3 ± 2.6 μg/mL) and was selected for further purification. From this fraction a bioactive compound was isolated and identified as the benzofuran euparin (EC50 = 0.47 ± 0.05 μg/mL). The EC50 values obtained for OE, fraction F2, and euparin demonstrated that a progressive increase in the anti PV-1 activity was gained during the purification process.This finding prompted us to perform a bioassay-guided fractionation of OE by chromatographic techniques. Among all the fractions obtained , and consequently high selectivity has been demonstrated with SI values of 284.9, 1068, and 854.7, respectively. These results prompted us to characterize the probable mechanism of action. As euparin showed neither virucidal activity nor protection of the cells in the pretreatment, we decided to evaluate which step of viral cycle was inhibited by this compound.Time of addition assays demonstrated that euparin completely inhibited the viral plaques formation when it was added in the postadsorption period. This result was later confirmed with time of addition assay on one-step replication viral cycle where we observed that the maximum inhibition was obtained when euparin was added during the first twenty minutes after infection. This inhibition was abolished when euparin was added beyond that time. These results suggest that the likely euparin mechanism of action would be the interference with one of the first events of poliovirus replication cycle probably on the penetration and/or uncoating process and not the viral RNA replication step. We could speculate that euparin exert a similar mechanism of action as other antiviral compounds called capsid binders as the WIN compounds . EuparinIn addition, the high selectivity activity against poliovirus in the submicromolar order, specially PV-1, and the specificity of euparin among the evaluated viruses make this an interesting compound for the development of new antipoliovirus drugs since many prototype drugs under clinical trials, as pleconaril, are unable to inhibit PV-1 .Eupatorium buniifolium and the isolation of euparin, as the active principle responsible for this activity. The results obtained contribute to describe a new biological activity in a known compound and suggest that euparin emerges as a new selective antipoliovirus drug or as a lead molecule for the developing of more potent derivatives. To our knowledge, this is the first report describing the presence of euparin in E. buniifolium and also the first detection of its antiviral activity. Further studies are being undertaken to define the molecular target of euparin.In this study we report the antipoliovirus activity of 1H-NMR (MeOH-d4) and 13C-NMR (DMSO-d6) spectra of euparin.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Saccharopolyspora rectivirgula, the causative agent of farmer’s lung disease. The draft genome consists of 182 contigs totaling 3,977,051 bp, with a GC content of 68.9%.We have sequenced the genome of  Saccharopolyspora rectivirgula is a Gram-positive thermophilic sporulating actinomycete  and Tris-EDTA (TE) buffer (10 mM Tris [pH 7.9] and 1 mM EDTA) using conditions previously described  platform. A total of 9.3 million paired-end reads were generated, with an average read length of 100 nucleotides. Assembly of the reads was done using the A5 assembly pipeline  (Saccharopolyspora spinosa (8.58 Mb)   and Sacc8.58 Mb) . By use 8.58 Mb) , it was AYJW00000000. The version described in this paper is the first version, AYJW01000000.This whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession no.


Deletion of the subtelomeric region of 1p36 is one of the most common subtelomeric deletion syndromes. In monosomy 1p36, the presence of obesity is poorly defined, and glucose metabolism deficiency is rarely reported. However, the presence of a typical Prader-Willi-like phenotype in patients with monosomy 1p36 is controversial.In this report, we describe two female patients, one who is 6 years 2 months of age and another who is 10 years 1 month of age, both referred to our hospital for obesity and a Prader-Willi-like phenotype. These patients presented with severe obesity , hyperphagia and developmental delay. Analysis of basal hormone levels showed normal thyroid function and adrenal function but considerable basal hyperinsulinism . In patient 1, glycaemia was 75 mg/dl (HOMA-R 10.09), and the HbA1c level was 6.1%; in patient 2, glycaemia was 122 mg/dl, and the HbA1c level was 6.6% (HOMA-R 14.82). An oral glucose tolerance test demonstrated impaired glucose tolerance and diabetes mellitus with marked insulin resistance .some patients with monosomy 1p36 may show Prader-Willi-like physical and physiologic characteristics such as obesity and hyperinsulinism with impaired glucose metabolism, which can cause type II diabetes mellitus. Further studies are necessary to evaluate these findings. Deletion of the subtelomeric region of 1p36 is one of the most common subtelomeric deletion syndromes, with an estimated incidence of 1 in 5000 to 1 in 10,000 live births .Monosomy 1p36 is responsible for a cluster of characteristic clinical features including mental retardation and multiple congenital abnormalities caused by haploinsufficiency of numerous contiguous genes ,6, and aIn monosomy 1p36, the occurrence of both obesity and or glucose metabolism disorders is rarely reported. However, the presence of a typical Prader-Willi-like phenotype in patients with 1p36 monosomy is controversial.In this report, we describe two female subjects with monosomy 1p36 and Prader-Willi-like phenotype who also exhibit type II diabetes and impaired glucose tolerance due to hyperinsulinism. We provide data both for and against this possible association. Written informed consent was obtained from the parents before publication of this case report and any accompanying images.A female patient, 6 years 2 months of age, was referred to our Paediatric Endocrinology Unit for severe obesity.I-7V-8X, and the infant required oral and gastric suctioning, as well as oxygen and stimulation upon delivery to treat cyanosis. Birth weight was 2.175 kg , length was 44 cm , and head circumference was 32 cm . The neonatal screening for congenital hypothyroidism was negative.The proposita was the third-born child (the first pregnancy was interrupted because of a disorder involving chromosome 7) of a nonconsanguineous marriage who was delivered normally at 40 weeks after an uncomplicated pregnancy. The family history was unremarkable, particularly for obesity, which was present only in a paternal uncle. The Apgar score was 5She presented with abnormal psychomotor development, as she sat upright at 8 months, began to walk independently at 15 months and began to use language at 15 months.th centile), her height was 110 cm , and her occipital-frontal circumference was 45.8 cm . This patient’s BMI was 26.4 , and the pubertal staging was defined as B1-PH2-3-AH2. Her blood pressure evaluation showed both systolic and diastolic hypertension (124/85 mmHg).At 6 yrs, 2 months of age, this patient weighed 32.000 kg , TSH , cortisol  and ACTH  values. The blood count was also normal. Her blood chemistry was AST: 45 IU/L, ALT: 35 IU/L, γ-GTP: 27 IU/L, HbA1c: 6.1%, TG: 132 mg/dL, total cholesterol: 195 mg/dL, HDL: 51 mg/dL, insulin: 54.5 μU/ml, glycaemia: 102 mg/dl (HOMA 13.72). A screening for coeliac disease was negative , but an oral glucose tolerance test indicated hyperinsulinism (peak insulin level was 297 μU/mL), impaired glucose tolerance (120’ glycaemia 179 mg/dl), and marked insulin resistance (120’ insulin level 193 μU/mL).Routine cytogenetic investigations revealed an apparently normal female karyotype . Chromosomal studies, also using FISH, ruled out a 15q11.2-q13 deletion. Tests for methylation and the presence of maternal uniparental disomy were both negative.Given the numerous clinical features observed in this subject, a FISH analysis utilising 41 subtelomere probes was performed ) using Vysis probes . The results were confirmed with a chromosome 1p subtelomere probe  and the D1Z2 midi-satellite probe with repeats in band 1p36 (Oncor), which is used to confirm a common deletion interval (1p36.3).Molecular karyotyping was performed by an array-CGH analysis using the proband's DNA and an Agilent 44 K array platform with a resolution of approximately 100 kb. Based on the physical mapping positions designated in the March 2006 assembly (NCBI36/hg18) of the UCSC Genome Browser, this analysis showed a deletion of approximately 1.5 Mb that involved the 1p36.33 region, with the breakpoint falling between 554,268 bp (first-deleted oligomer) and 2,133,973 bp (last-deleted oligomer) , her length was 44.6 cm , and her head circumference was 32 cm . Central hypotonia occurred soon after birth. The screening for neonatal congenital hypothyroidism was negative, but she experienced recurrent respiratory infections throughout the first year of life.The Apgar score was 6At 6 months of age, persistence of patent ductus arteriosus (PDA), which lead to pulmonary hypertension, was diagnosed; PDA was closed at 7 months. She presented with abnormal psychomotor development, as she began to sit upright at 9 months, walk independently at 17 months and demonstrated use of language at 19 months. Bilateral neurosensory deafness was diagnosed at 1 year of age. The child developed significant hyperphagia.th centile), her length was 131.8 cm , and her BMI was 27.7 . The pubertal staging was defined as B1-PH2-3-AH2. Blood pressure evaluation showed systolic and diastolic hypertension (124/85 mmHg).At 10 yrs 1 month of age, the proposita showed a phenotype consistent with Prader-Willi syndrome: the patient weighed 52.600 kg , TSH , cortisol  and ACTH 14.1 (range 9.0-52.0 ng/L) values. The blood count was also normal, and her blood chemistry was as follows: AST: 20 IU/L, ALT: 21 IU/L, γ-GTP: 32 IU/L, HbA1c: 5.5%, TG: 146 mg/dL, total cholesterol: 259 mg/dL, HDL: 45 mg/dL, prolactin: 271 mIU/L, HbA1c: 6.6%, insulin: 49.2 μU/ml, glycemia 122 mg/dl (HOMA-R 14.82). The screening for coeliac disease was negative , and an oral glucose tolerance test indicated diabetes mellitus (120’ glycaemia 234 mg/dl), with marked hyperinsulinism and insulin resistance .Routine cytogenetic investigations revealed an apparently normal female karyotype . Chromosomal studies, which also used FISH, ruled out a 15q11.2-q13 deletion found in Prader-Willi syndrome. The hypophyseal RMN revealed an empty sella.rd centile), BMI was 29.9 , and pubertal staging was described as B2-3-PH4-AH2. Menarche started at 13 years of age.At 12 yrs, 8 months, her height was 141 cm , her weight was 56.000 kg , and pubertal staging was found to be B5-PH5-AH3.At 16 years of age, her height was 145 cm , using Vysis probes . The results were confirmed with a chromosome 1p subtelomere probe  and the D1Z2 midi-satellite probe with repeats in band 1p36 (Oncor). The D1Z2 probe is used for the confirmation of a common deletion interval (1p36.3). Array-CGH was performed using the Agilent Human Genome CGH microarray kit 180 K, with a resolution of approximately 40 kb. Break point positions were reported according to Hg19, with build 37 falling between 564,424 bp (first-deleted oligomer) and 3,111,905 bp (last-deleted oligomer), which showed a deletion of approximately 2.5 Mb that involved the 1p36.33 – 1p36.32 region , cardiovascular malformations , central nervous system defects , neonatal hypotonia, cortical dysplasia, and seizures ,9,10. Afet al.[In patients with monosomy 1p36, the presence of obesity has been poorly defined, and glucose metabolism disorders have been infrequently reported to date. For example, D'Angelo et al. identifiOur case reports confirm this association and suggest that this phenotype may be associated with obesity in some 1p36 patients. We also present evidence that hyperinsulinism and impaired glucose tolerance or diabetes mellitus may be present in these patients and that these features can occur in younger patients.In fact, genome-wide linkage studies suggest that the 1p36 region shows a strong correlation with several traits normally associated with metabolic syndrome (MetS) , such asPrader-Willi-like phenotype has been described in patients with chromosomal duplications ,16, FragObesity and/or hyperphagia are features rarely described in patients with 1p 36 deletion ,21-24, bTo help paediatric neurologists and other professionals in the recognition of this emerging and common chromosomal syndrome and to identify the possible overlap of clinical presentations between patients with 1p36 deletion syndrome and patients with a Prader-Willi like phenotype, we have reviewed published articles that describe patients with these features.et al. [et al.[et al.[D’angelo et al.  describe. [et al. reviewedl.[et al. describel.[et al..Thus far, no individual gene within this region has been conclusively determined to be causative of any component of the phenotype . Howeveret al.[Interestingly, D’Angelo et al. have fouet al.. This maet al..Approximately 90% of the 1p rearrangements are less than 10 Mb in size , and it In conclusion, our case reports confirm that some patients with 1p36 monosomy may show a Prader-Willi-like physical phenotype and that this aspect may therefore be overlooked, which may result in this condition being underdiagnosed. These patients may show such characteristics as obesity with hyperinsulinism and impaired glucose metabolism. Further studies are necessary to further evaluate this aspect.Written informed consent was obtained from the parents of the patients for publication of this case report and any accompanying images.The authors declare that they have no competing interests.SStagi carried out the endocrinological evaluation, conceived of the study and participated in its design. EL performed the clinical genetic diagnosis. SSeminara performed the endocrinological evaluation. MP performed the molecular genetic studies. FC participated in the endocrinological evaluation. MdM participated in the endocrinological evaluation and participated in its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/15/16/prepub


A new method based on cloud-point extraction has been developed, optimized and validated for the determination of ergosterol in rat plasma, urine and faeces by liquid chromatography. The non-ionic surfactant Triton X-114 was chosen as the extract solvent. The chromatographic separation was performed on an Inertsil ODS-3 analytical column with a mobile phase consisting of methanol and water  at a flow rate of 1 mL/min. The methodology was validated completely. The results indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. The method was successfully applied to the pharmacokinetic studies of ergosterol in rats. The results indicate that the ergosterol levels in feces are much higher than those in plasma and urine of the rat.Ergosterol from many medicinal fungi has been demonstrated to possess a variety of pharmacological activities  Polyporus umbellatus, Cordyceps sinensis, and Hypsizigus marmoreus [Polyporus umbellatus [in vivo might be influenced by many factors, such as body-and/or cell-compartment distribution, drug metabolism, lipophilicity, membrane permeability, and protein binding. So, these multiple pharmacological activities of ergosterol make it worth carrying out further study on pharmacokinetic properties and elimination pathway of ergosterol.Ergosterol is one of the best-known steroids, which exists widely in many medicinal fungi such as armoreus –3. We habellatus . Ergostebellatus  and antiA number of methods have been reported for the quantification of ergosterol in raw materials, such as high performance liquid chromatography-ultraviolet detection (HPLC-UV) , 6–11, hPolyporus umbellatus. The procedure isolation and purification of ergosterol was published in our previous paper [1H NMR, 13C NMR, and MS) and compared with those found in the literature [Polyporus umbellatus purified in our laboratory with 99% purity as determined by HPLC. HPLC-grade methanol was purchased from Baker Company . Ultrahigh purity water was prepared by a Millipore-Q SAS 67120 MOLSHEIM (France).The standard of ergosterol  was isolus paper . Its strterature . Ergostaµg/mL) and ergone  were prepared by dissolving suitable amounts of pure substance in acetone and were stored in darkness at 4°C.Standard stock solutions of ergosterol  samples were separately prepared in a similar manner as those used for the calibration curve. Concentrations of 0.25, 0.75, and 2.00 µg/mL were used for plasma and urine calibration standards, whereas concentrations of 0.25, 25, and 100 µg/mL were used for faecal homogenate corresponding to the low QC, medium QC, and high QC, respectively.Faecal samples were homogenized with acetone in the ratio 1 : 10 (g : vol = faeces : acetone) to obtain faecal homogenate. For HPLC determination, calibration standards of ergosterol at concentration levels of 0.09, 0.25, 0.50, 0.75, 1.00, 2.00, and 2.50 µm, Japan) with the column temperature set at 30°C. An isocratic elution of methanol (A) and water (B) was 98 : 2 (v/v) in 23 min. The flow rate was 1.0 mL/min with detector wavelength set at 283 nm (maximum absorption wavelength of ergosterol), and the injection volume was 20 µL.HPLC was performed with a Waters 2695 instrument composed of a quaternary pump, a column oven, and a Waters 2487 dual wavelength absorbance detector, and Empower was used for data collection. The chromatographic separation was performed on an Inertsil ODS-3 analytical column  of IS working solution and 135 µL of 0.3 mol/L sodium chloride solution. The contents were mixed for 2 min, and then followed by the addition of 100 µL of 5% Triton X-114 (v/v) aqueous solution. After that, the obtained contents were well mixed again for 5 min and then incubated in the thermostatic bath at 40°C for 20 min. After the phase separation was formed by centrifugation at 5000 rpm for 10 min, the surfactant-rich phase was obtained in the bottom of the tube by the removal of the water phase. 100 µL of mobile phase was spiked to the surfactant-rich phase. Then, the contents were mixed and centrifuged at 16,000 rpm for 5 min, respectively. Most of the surfactants and coextractants such as hydrophobic proteins were precipitated in the bottom of the tube, and 20 µL of supernatant fluid was injected into the HPLC system for analysis.The plasma or urine was prepared by a cloud-point extraction method. In a 1.5 mL capped centrifugal tube, 50 µL volume of faecal homogenate standard or sample was transferred to a 1.5 mL centrifuge tube, and then 30 µL of IS working solution was spiked and vortex-mixed for 1 min. 70 µL of acetone was added to the solution and vortex again for 2 min. The mixture was centrifuged at 10,000 ×g for 10 min. A 20 µL of supernatant of the solution was injected into the HPLC for analysis.A 100 µg/mL) and calibration standards for faecal homogenate  were extracted and assayed. The linearity of the calibration curve was confirmed by plotting the peak-area ratios of ergosterol to IS versus the ergosterol concentrations with a 1/x-weighted least-squares linear regression analysis. The limit of detection (LOD) was considered as the final concentration producing a signal-to-noise ratio of 3. The lower limit of quantification (LLOQ) was considered as the lowest concentration on the calibration curve where precision was within 20% and accuracy was within 100 ± 20% [ HPLC-UV method was applied for the quantification of ergosterol from rat plasma, urine, and faeces. Calibration standards of seven concentrations of ergosterol for plasma and urine  thaw (room temperature) cycles.To validate the method with real samples, a trial was undertaken to determine ergosterol in the plasma and excretion samples of healthy rats, which were administered a single dose of ergosterol orally. The study was conducted in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China. All procedures and the care of the rats were in accordance with institutional guidelines for animal use in research. Rats were housed in individual metabolic cages on standard laboratory food bedding in a temperature controlled room (22 ± 2°C) with a 12 h light/dark cycle. n = 6) based on the time of blood sampling with two animals each. The control groups received the vehicle only. The blood samples (approximately 300 µL) were collected from vena orbitalis in heparinized tubes at control and 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 24, and 36 h after the administration. Samples were immediately centrifuged at 5,000 ×g for 10 min and the plasma was frozen at −20°C and stored until analysis. The estimation of ergosterol in all the samples was undertaken within 36 h of blood collection by the method described previously. Male Sprague-Dawley (SD) rats, 200 ± 10 g, fasted overnight with free access to water for at least 12 h, were dosed orally by gavages with 100 mg/kg body weight of ergosterol dissolved in plant oil as a vehicle. Rats were divided into three groups (n = 6) based on the time of urine and faeces sampling with three animals each. The control groups received the vehicle only. Rats were housed with free access to food and water in individual metabolic cages, except for the final 12 h before a single oral administration of 100 mg/kg of ergosterol (access to water was ad libitum during the experiment). Faeces and urine were collected after administration in different periods . The amount of faeces and urine collected over each period was recorded, respectively, and then urine and faeces were stored at −20°C until analysis.Excretion was studied in another four groups  and pharmaceutical parameters were obtained.The percentage of ergosterol eliminated in faeces was calculated with the accumulated ergosterol eliminated in all periods divided by administration amount .1H NMR  3.63 , 5.39 , 5.58 , 0.63 , 0.95 , 1.04 , 5.18 , 5.22 , 0.83 , 0.84 , 0.92 ; 13C NMR  38.3 (C-1), 32.1 (C-2), 70.6 (C-3), 40.8 (C-4), 139.9 (C-5), 119.7 (C-6), 116.4 (C-7), 141.5 (C-8), 46.4 (C-9), 37.1 (C-10), 21.3 (C-11), 39.0 (C-12), 42.8 (C-13), 54.7 (C-14), 23.1 (C-15), 28.4 (C-16), 55.8 (C-17), 12.2 (C-18), 17.7 (C-19), 40.5 (C-20), 21.2 (C-21), 135.5 (C-22), 132.1 (C-23), 42.9 (C-24), 33.2 (C-25), 19.8 (C-26), 20.1 (C-27), 16.4 (C-28). The data of mass spectrum was published in our previous paper [The characteristics of ergosterol are  CDCl3, 1 MHz 38.3µm or Diamonsil C18 column, 250 mm × 4.6 mm i.d., 5 µm), temperature (30 or 35°C), and peak shape. The test results showed that the peak shapes of ergosterol were improved by the solvent system of methanol and water.Chromatographic optimisation of HPLC conditions was carried out with respect to mobile phase conditions (methanol-water or acetonitrile-water), stationary phase  from blank rats, each as a blank sample and a spiked sample. The chromatograms of ergosterol and the IS are showed in Figures µg/mL for plasma and urine and 0.08–125 µg/mL for faecal homogenate. The typical equation was Y = 0.8251  X + 0.0357 (r = 0.9996) for plasma, Y = 2.9189  X + 0.1265 (r = 0.9995) for urine, and Y = 0.0262  X + 0.0104 (r = 0.9994) for faecal homogenate. Y = peak-area ratio (ergosterol/IS) and X = ergosterol concentration. During routine analysis, each analytical run included a set of calibration samples, a set of QC samples, and the unknowns. The LOD of ergosterol in rat plasma, urine, and faecal homogenate were 42, 50, and 49 ng/mL, respectively. The LOQ of ergosterol in rat plasma, urine, and faecal homogenate was 90, 90, and 80 ng/mL respectively.Calibration curves of ergosterol were linear over the concentration range of 0.09–2.50 The selectivity of the method was demonstrated by comparing chromatograms of five independent biological samples  from blank rats, each as a blank sample and a spiked sample. n = 5). Recovery data are shown in The mean extraction recovery of ergosterol from plasma, urine, and faecal homogenate was 90.9 ± 2.9%, 92.4 ± 2.6%, and 93.7 ± 3.3%, respectively. The mean relative recovery for IS was 94.5 ± 3.2%  made this method suitable for routine analysis in preclinical pharmacokinetic studies, and the method was helpful in clinical pharmacokinetic studies. It can also be used as a reference for therapeutic drug monitoring of ergosterol.


N,N’’-dimethyldiethylene triamine (DMDETA), has been synthesized and evaluated for sub-mol % copper-catalyzed C–N cross-coupling reactions. The efficiency of the ligand was determined by kinetic methods. DMDETA proved to display efficiency similar to DMEDA and, in addition, the resulting catalyst was tolerant to air.A new efficient ligand,  Copper-catalyzed heteroatom cross-coupling reactions  are important synthetic tools in modern organic synthesis –6. Even 4, as well as BOC-protection followed by reduction by LiAlH4, were also evaluated as potential synthetic routes, but both methods proved unsuccessful. In our hands, protecting all nitrogens followed by methylation of the two remaining free valencies on N was the most efficient method for synthesizing DMDETA.Although DMEDA has proved to be a very efficient ligand for C–N, C–O and C–S, some issues still remain, such as the sensitivity to air. As the Me–NH–ethylene–NH motif seemed to be necessary when lowering the copper concentration, dimethyldiethylene triamine (DMDETA) was a natural choice as ligand. The ligand was synthesized based on literature procedures starting from diethylene triamine  85–86].–86.85–863 did solve the problem, but at the expense of lower yield. Distillation over CaH2 gave the pure DMDETA (4) in moderate yield.The removal of the tosyl groups proved to be complicated due to issues with isolation of DMDETA during the workup procedure. Steam-distillation followed by acidification by HCl (aq) to produce the DMDETA trihydrochloride-salt To test the efficiency of DMDETA a kinetic study was performed by varying the concentration of the ligand and copper. As a standard reaction the copper-catalyzed cross coupling of pyrazole with iodobenzene was chosen .The kinetic profile when varying the concentration of DMDETA proved to be more or less the same as for our previously reported results concerning DMEDA  77]77]The reaction order in copper was determined to be 0.7 . This isSince DMDETA seems superior to DMEDA in solubilizing the catalyst and hence generating a more stable active catalyst, we decided to challenge the reaction with conditions where DMEDA fails, by testing the reaction without an inert atmosphere. Somewhat to our surprise, we found that the DMDETA-based catalyst was efficient even under air .As can be seen from None of the ligands in In summary, a new and efficient ligand (DMDETA) for sub-mol % copper-catalyzed C–N cross coupling has been synthesized and evaluated in kinetic studies. DMDETA, together with DMEDA, is one of the few reported ligands that work at sub-mol % levels of copper catalyst ,77–7888.78General: All the experiments were carried out under inert atmosphere (nitrogen). DMEDA, iodobenzene and dodecane were distilled over calcium hydride. Toluene was dried through distillation and stored under nitrogen. DMF was distilled over calcium hydride and stored under nitrogen. CuCl2 , K3PO4 (98%), and pyrazole (98%) were stored under air- and moisture-free conditions. All other chemicals were used as received. A gas chromatograph with flame ionization detector and a 30 m × 0.25 mm EQUITY-5 fused silica capillary column was used, with hydrogen as carrier gas. General temperature program: 100 °C for 14 min, then up to 300 °C at 50 °C min−1 for 2 min. Dodecane was used as an internal standard. The NMR spectroscopic analysis was carried out on a Varian 400 MHz instrument by using CDCl3 or D2O as solvent, with shifts measured against TMS.4-Methyl-N,N-bis(2-(4-methylphenylsulfonamido)ethyl)benzenesulfonamide (1): Diethylene triamine  and NaOH  were added to a round-bottom flask, and H2O was added under stirring until the hydroxide was dissolved. Tosyl chloride  was dissolved in DEE (300 mL) and added over 1 h to the reaction mixture, followed by stirring at ambient temperature for 4 h. The resulting white solid was collected by filtration, washed repeatedly with hot water, and then with EtOH (3 × 50 mL), and dried in vacuo to yield 1 . 1H NMR  δ 2.43 , 3.16 , 7.31–7.77  ppm; 13C NMR  δ 21.55 (3C), 42.65 (2C), 50.56 (2C), 127.16–127.31 (6C), 129.83–130.00 (6C), 134.67 (2C), 136.63 (3C), 143.65 (1C) ppm.N,N-bisethyl)-4-methylbenzenesulfonamide (2): 1  was dissolved in dry DMF (500 mL) whereupon NaH  was added in portions under stirring, and stirring was then continued for 30 min at ambient temperature. Excess NaH was separated by allowing the solids to settle, whereupon the solution was removed with a syringe. The solution was heated to 120 °C. Iodomethane  was added slowly under stirring at 120 °C and then heated under reflux for 24 h. Half of the solvent was evaporated, whereupon water was added (400 mL), resulting in a white/yellow precipitation. The solids were collected by filtration, washed repeatedly with hot water, and dried in vacuo to yield 2 . 1H NMR  δ 2.43 , 2.78 , 3.29 , 7.33 , 7.69  ppm; 13C NMR  δ 21.53 (3C), 36.28 (2C), 48.22 (2C), 49.74 (2C), 127.40–127.51 (6C), 129.82–129.91 (6C), 133.94 (2C), 135.30 (3C), 143.61–143.87 (1C) ppm.N,N’’-dimethyldiethylene triamine trihydrochloride (3): 2  was dissolved in conc. H2SO4 (90 mL) under heating, with continued stirring at 120 °C for 18 h. A saturated KOH/H2O solution was added slowly to the reaction mixture (cooled on ice) until it was strongly alkaline. The mixture was then steam-distilled, and the distillate was acidified with HCl and evaporated to dryness to yield the HCl salt of dimethyldiethylene triamine . The salt was further purified by heating under reflux in EtOH (99.8%), and, upon cooling on ice, was filtrated and washed with ice cold EtOH to give the pure HCl-salt of N,N’’-dimethyldiethylene triamine (3) . 1H NMR  δ 2.69 , 3.38  ppm; 13C NMR  δ 33.23 (2C), 43.32 (2C), 44.06 (2C) ppm.N,N’’-dimethyldiethylene triamine (4): The HCl-salt 3 was distilled over CaH2 by Kugelrohr distillation, at 160–200 °C under vacuum, to yield a clear oil of 4 (40–60%). 1H NMR  δ 1.22 , 2.39 , 2.68  ppm; 13C NMR  δ 36.45 (2C), 49.27 (2C), 51.71 (2C) ppm.General procedure for the kinetic experiments: The initial rate log plots were constructed from data points ranging from 0 to a maximum of 25% yield. The kinetic investigation was carried out by varying the concentration of one component and keeping the rest of the components constant under standard reaction conditions; pyrazole , iodobenzene , CuCl2  K3PO4 , DMDETA  and dodecane . Into a microwave vial was added pyrazole (A mmol) and K3PO4 (B mmol). The vial was sealed and a CuCl2 solution  was added. The THF was removed by three cycles of vacuum followed by nitrogen, whereupon toluene (2 mL), DMEDA (D mmol), iodobenzene (E mmol), and dodecane  were added. The closed vial was placed in a preheated aluminum block at 135 °C. Samples (50 μL) were collected at intervals, filtered through a small silica plug, and analyzed by GC. The yield from the GC was determined by using dodecane as the internal standard.Procedure for running under air or nitrogen: The same procedure as the kinetic experiment was used; however, the reactions were not run in closed vials but under reflux in air or nitrogen. In this case 20 mol % of the ligands were used, DMEDA  and DMDETA . Samples were taken (50 μL) at certain time intervals by using the same workup procedure as mentioned above. The yield from the GC was determined by using dodecane as the internal standard.Procedure for ligand screening: The same procedure as the kinetic experiment was used. 20 mol % of triethylene tetraamine , tetraethylene pentaamine , pentaethylene hexamine , polyethyleneimine (MW ≈ 800) , StratoSphere™ PL-EDA resin , StratoSphere™ PL-DETA , tris(2-aminoethyl)amine polymer-bound . Samples (50 μL) were taken after 24 h by using the same workup procedure as previously mentioned. The yield from the GC was determined by using dodecane as the internal standard.File 11H NMR and 13C NMR data of compounds 1–4.


Aliihoeflea sp. strain 2WW, which consists of a 4.15-Mb chromosome and contains different genes that are involved in arsenic transformations.Here, we report the draft genome sequence of the arsenite-oxidizing bacterium  Aliihoeflea sp. strain 2WW was isolated from a biofilter treating arsenic-contaminated groundwater in the city of Cremona . It is a strictly aerobic, mobile, and rod-shaped Gram-negative bacterium. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain is closely (98.7%) affiliated with Aliihoeflea aestuarii N8T ] and arsenite [As(III)], respectively. Under heterotrophic conditions, it can oxidize As(III) to As(V), with specific growth rates (h-1) of 0.23, 0.13, and 0.006 at 30°C, 15°C, and 5°C, respectively. In growth experiments, As(III) was always oxidized to As(V) in the early exponential growth phase and the oxidation was complete within 24 h at 30°C, 96 h at 15°C, and 350 h at 5°C. Complete As(III) oxidation occurred in the pH range 5.0 to 8.0. The resting cells of an As(III)-induced culture of strain 2WW were able to oxidize completely 200 µg liter-1 of As(III) in 8 h, while noninduced cells oxidized As(III) in 24 h.Aliihoeflea sp. strain 2WW was obtained by Illumina sequencing , producing paired-end and mate-pair sequence reads of ~50 bp, with insert sizes of ~300 bp and ~4,000 bp, respectively. CLC Genomics Workbench version 5.5.1  was used to assemble the quality-filtered paired-end reads into 273 contigs. Subsequently, SSPACE Premium scaffolder version 2.0 ], and the second containing an arsenite oxidase, aioBA. Phylogenetic analysis of aioA confirmed the phylogenetic affiliation with the Alphaproteobacteria and showed close affiliation with members of the family Phyllobacteriaceae, although 2WW is not chemoautotrophic. Although 2WW possesses arsC, normally retrieved in the ars operon of As(V)-resistant strains, it was not able to reduce As(V) to As(III). In addition, many genes encoding putative metal  transporters and cobalt-zinc-cadmium resistance protein (CzcD) were also identified on the genome.The genome sequence of Aliihoeflea sp. strain 2WW has been deposited in GenBank under the accession no. AYOD00000000.The draft genome sequence of


Background. Maternal fever during labor epidural analgesia (LEA) may cause increased maternal and cord serum inflammatory cytokines. We report the effects of intermittent and continuous LEA on these cytokines. Methods. Ninety-two women were randomly assigned to continuous (CLEA) or intermittent (ILEA) groups, 46 in each. Maternal temperature was checked and blood drawn at epidural insertion (baseline) and four-hourly until 4 h postpartum (4 PP). Cord blood was drawn after placental delivery. Interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte macrophage-colony stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) were measured and analyzed according to group randomization, and then combined and reanalyzed as febrile (temperature ≥38°C) or afebrile groups. Results. Significant intragroup changes from baseline were noted in some groups. Data are pg/mL, median (Q1/Q3). IL-6 rose at all time points in all groups. CLEA: baseline: 18.5 (12.5/31.1), 4 h: 80.0 (46.3/110.8), 8 h: 171.9 (145.3/234.3), and 4 PP: 81 (55.7/137.4). ILEA: baseline: 15.7 (10.2/27.1), 4 h: 68.2 (33.3/95.0), 8 h: 125.0 (86.3/195.0), and 4 PP: 70.2 (54.8/103.6). Febrile group: baseline: 21.6 (13.8/40.9), 4 h: 83.9 (47.5/120.8), 8 h: 186.7 (149.6/349.9), and 4 PP: 105.8 (65.7/158.8). Afebrile group: baseline: 10.9 (2.1/17.4), 4 h: 38.2 (15.0/68.2), 8 h: 93.8 (57.1/135.7), and 4 PP: 52.9 (25.1/78). IL-8 rose at all time points in CLEA: baseline: 2.68 (0.0/4.3), 4 h: 3.7 (0.0/6.5), 8 h: 6.0 (3.3/9.6), 4 PP: 5.6 (0.8/8.0), and afebrile group baseline: 2.5 (0.0/4.7), 4 h: 3.3 (0.0/6.2), 8 h: 5.3 (1.9/9.8), and 4 PP: 4.7 (0.0/7.6). It fell at 4 PP in febrile group: baseline: 4.1 (0.0/6.4), 4 h: 3.8 (0.0/6.5), 8 h: 5.2 (2.5/8.0), and 4 PP: 2.9 (0.0/4.0). GM-CSF increased at 8 h and decreased at 4 PP in ILEA baseline: 2.73 (0.0/7.2), 4 h: 2.73 (0.0/7.9), 8 h: 3.9 (2.7/11.5), and 4 PP: 2.0 (0.0/7.2). It increased at 4 h and 8 h and decreased at 4 PP in febrile group: baseline: 2.6 (0.0/4.2), 4 h: 3.2 (2.1/7.0), 8 h: 4.0 (3.2/12.3), and 4 PP: 2.4 (1.7/12.6). There were no intergroup cytokine changes in maternal or cord serum in CLEA versus ILEA or febrile versus afebrile groups. Conclusions. Some cytokines, especially IL-6, rise physiologically during labor epidural analgesia. Maternal intrapartum fever may occur with labor epidural analgesia (LEA). The mecWe previously reported that intermittent, versus continuous, LEA had a lower incidence of maternal and neonatal fever in the first few hours of epidural analgesia initiation . In the After Institutional Review Board (IRB) approval, 92 women gave informed consent and were enrolled in this prospective, randomized unblinded study. As required by our IRB, a parent or legal guardian provided consent for enrolled minors. A computer-generated program  randomized an equal number of women (46) to receive continuous or intermittent lumbar epidural analgesia . Inclusion criteria were a healthy, nulliparous parturient in spontaneous labor with a term, singleton fetus in vertex presentation. Exclusion criteria were a baseline temperature of ≥37.5°C, suspected chorioamnionitis, history of drug abuse, and pregnancy-related complications such as preeclampsia, intrauterine growth retardation, and gestational diabetes mellitus.μg and 8 mL of an analgesic solution: either 0.125% bupivacaine with fentanyl 0.0002% or 0.1% ropivacaine with fentanyl 0.0002%. In the CLEA group, a continuous infusion of one of the analgesic solutions was administered at 10–15 mL/hr with a goal of maintaining a T-10 or higher sensory blockade. In the ILEA group, 10–15 mL of the solution was given by manual injections as requested by the patient. The labor and delivery rooms were maintained at a temperature comfortable for the patient, generally between 20 and 22°C.One of the investigators initiated the epidural analgesia at maternal request, using strict sterile precautions . In all patients, LEA was initiated with a 3 mL test dose of 1.5% lidocaine with 1 : 200,000 epinephrine, followed by fentanyl 100  Baseline measurements noted at initiation of epidural analgesia included maternal age, height, weight, gestational age, cervical dilatation, and tympanic temperature. Peripheral venous blood (5 to 10 mL) was also drawn at this time for cytokine analysis. The temperature measurement and venipuncture were repeated at four-hourly intervals, with the final sampling occurring four hours postpartum (4 PP). Following fetal and placental delivery, umbilical vein cord blood was collected for cytokine analysis. A pediatric nurse measured the neonatal rectal temperature within 30 minutes of delivery.β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α). The sensitivity of the test for IL-1β, IL-6, IL-8, GM-CSF, and TNF-α was 15 pg/mL, 3 pg/mL, 3 pg/mL, 15 pg/mL, and 10 pg/mL, respectively. The respective intra- and inter-assay precision ) for the different cytokines was: IL-1β: 1410 (117) and 1440 (127), IL-6: 883 (58) and 892 (62), IL-8: 1020 (73) and 987 (97), TNF-α: 1130 (92) and 1130 (94), and GM-CSF: 2137 (75) and 2084 (98).The blood samples were collected in serum separation tubes and refrigerated immediately. Within 24 hours, the serum was separated by centrifugation at 2500 g for 20 minutes . The serum was then placed in duplicate aliquots and immediately frozen and stored at −70°C until analysis. The samples were batched in groups of 50–90 samples for laboratory analysis. They were analyzed within 3 months of collection, by a Multiplex Bead Immunoassay technique . The serum cytokine levels assayed were interleukin-1B streptococcal colonization of the genital tract, times of membrane rupture and full cervical dilatation, time and type of delivery, epidural insertion to full dilatation and delivery, and neonatal sepsis evaluation rate were collected. At our institution, clinical chorioamnionitis is diagnosed on the basis of maternal fever (temperature ≥38°C) plus one or more of the following: maternal or fetal tachycardia, uterine tenderness, foul smelling amniotic fluid, or maternal leukocytosis [Labor and epidural characteristics like the number of vaginal examinations, use of internal monitors or oxytocin augmentation, clinical suspicion of chorioamnionitis, group ocytosis . ClinicaA primary analysis between the two groups was performed. The two groups were then combined, and a reanalysis made between “febrile” and “afebrile” groups to look for any association between cytokines and the development of fever. Parturients who developed intrapartum fever (temperature ≥38°C) were classified as the febrile group and the rest as the afebrile group.t-test. Distributions of cytokine levels were markedly skewed; thus nonparametric methods were used for the cytokine investigations. Tests of changes in cytokine levels over time, and comparisons of those changes by infusion method and presence of fever were analyzed using the Wilcoxon sign and sign-rank tests. For statistical significance, a more stringent threshold of P < 0.01 was used to control for multiple comparisons. This was based on the false discovery rate method [Demographic data were analyzed by Student's e method .The sample size was determined for our original study based on the assumed incidence of fever with CLEA and ILEA .No technical problems or complications were encountered during the insertion or maintenance of epidural analgesia. There were no differences between the CLEA and ILEA groups in demographic and baseline data  .P = 0.036). At 8 h and thereafter, no differences were noted. The overall incidence of fever was 14/46 (30.4%) in CLEA and 12/43 (27.9%) in ILEA (P = ns). Regrouping based on the development of fever resulted in 26 women falling into the febrile group and 66 into the afebrile group. Highly significant differences were seen between these two groups at all time points  at all time points in all groups—CLEA, ILEA, febrile and afebrile. IL-8 also showed a highly significant rise (P ≤ 0.0075) at all time points in CLEA and afebrile groups; in the febrile group, it showed a fall at 4 PP, while in ILEA there was no change.GM-CSF increased at 8 h and decreased at 4 PP in ILEA; in the afebrile group, it increased at 4 h and 8 h and decreased at 4 PP, while in CLEA and febrile groups there was no change. There was no change in IL-1β or TNF-α at any time point in any group. Intergroup analysis of CLEA versus ILEA and febrile versus afebrile groups showed no significant difference in any cytokine at any time point.Intragroup and intergroup analysis of changes from baseline is shown by infusion type in There were no significant differences between CLEA and ILEA. Although neonates born to febrile versus afebrile mothers had higher temperatures , there wThe labor and epidural characteristics of CLEA and ILEA were shown in our previous publication , and thoTo our knowledge, the effect of intermittent versus continuous LEA on maternal and cord serum cytokine expression has not been previously reported.Our data indicate that some maternal serum cytokines increase with LEA. IL-6 exhibited the most consistent intragroup change, increasing during labor and remaining elevated for at least 4 h postpartum, regardless of CLEA or ILEA and febrile or afebrile group status. There were no CLEA/ILEA or febrile/afebrile intergroup differences in maternal or cord sera. Thus, it is interesting to note that at the 4 h time point, a significantly higher incidence of fever in CLEA versus ILEA group yielded no differences in IL-6 levels. Thus, maternal and cord IL-6 levels appear independent of the method of epidural analgesia, the development of fever, and neonatal temperature.Our findings differ from some studies that have evaluated maternal serum IL-6 levels and intrapartum fever. Goetzl et al. , whose pRise of maternal serum cytokines, particularly IL-6 may be a normal response to labor –13. ArntSerum IL-6 levels also physiologically increase in response to high-intensity exercise in humans –19, whicWe therefore do not think that a rise in IL-6 represents a pathological response. Our study certainly found that to be true.β) was either undetected [α or GM-CSF during labor.We found statistically significant intragroup changes in other cytokines. Clinical significance of these findings is unclear. Our findings have similarities and differences from other studies in the obstetric population. In the Goetzl et al.  study, Idetected  or no chdetected , 9, 13, detected , 12. TheIn contrast to our study, Smulian et al. and GoetzLimitations in our study include the use of different local anesthetics (ropivacaine and bupivacaine), which may have confounded our results. However, as discussed in our paper that studied the same patients , we founAnother limitation is that we did not have a control group that did not receive epidural analgesia. Such a study would be difficult at our institution, where approximately 90% of laboring women receive epidural analgesia.In conclusion, we found that maternal serum inflammatory cytokines rise during LEA, irrespective of the method of epidural dosing (intermittent or continuous) or the development of fever. These findings suggest that a rise in maternal serum cytokines during LEA may be physiological, and that there may be no pathological basis for epidural fever based on proinflammatory cytokine analysis.


Streptomyces xiamenensis strain 318, shows multiple biological effects, including anti-fibrotic and anti-hypertrophic scar properties. To increase the diversity in the structures of the available benzopyrans, by means of biosynthesis, the strain was screened for spontaneous rifampicin resistance (Rif), and a mutated rpsL gene to confer streptomycin resistance (Str), was introduced into the S. xiamenensis strain M1-94P that originated from deep-sea sediments. Two new benzopyran derivatives, named xiamenmycin C (1) and D (2), were isolated from the crude extracts of a selected Str-Rif double mutant (M6) of M1-94P. The structures of 1 and 2 were identified by analyzing extensive spectroscopic data. Compounds 1 and 2 both inhibit the proliferation of human lung fibroblasts (WI26), and 1 exhibits better anti-fibrotic activity than xiamenmycin. Our study presents the novel bioactive compounds isolated from S. xiamenensis mutant strain M6 constructed by ribosome engineering, which could be a useful approach in the discovery of new anti-fibrotic compounds.The benzopyran compound obtained by cultivating a mangrove-derived strain,  During daily urban life, a causal relationship between the elevated urban air pollution and an increased severity of airway diseases, including lung fibrosis and lung cancers, has been supported by epidemiological and toxicological research ,2. FibroN--2H-1-benzopyran-6-yl)carbonyl)-threonine, was obtained by cultivating the mangrove-derived strain Streptomyces xiamenensis 318, and it was found to have multiple biological effects toward inhibiting fibrosis, i.e., the inhibition of excessive lung fibrosis in vitro and the attenuation of hypertrophic scars by the suppression of local inflammation, and by the reduction of the effects of mechanical stress + peak at m/z = 406.2210 by HRESIMS, consistent with an elemental composition of C22H31NO6, which was 15 amu more than the molecular weight of xiamenmycin. The 1H and 13C NMR data showed an additional methyl group due to the presence of signals at δH 3.65  and δC 52.3 (CH3). The HMBC correlations between CH3-7′ and CO-6′ (δC 171.8) confirmed the methyl esterification. The good agreement of the respective NMR data of 2 with those of xiamenmycin, together with ROESY data and proton coupling constants indicated that both compounds shared the same relative configurations of pyran-ring and the amino acid portion. Compound 2 possessed the same stereo-configurations as xiamenmycin, confirmed by the similar CD spectrum. Therefore, the structure of 2 was determined as -methyl 3-hydroxy-2--3-hydroxy-2-methyl-2-(4-methylpent-3-enyl)chroman-6-carboxamido)butanoate, named xiamenmycin D.Compound 1 and 2 against human lung fibroblast. WI26 cells were exposed to 1 at a concentration of 15 μg/mL, 2 at a concentration of 30 μg/mL, and xiamenmycin at a concentration of 30 μg/mL, respectively, for 0, 1, 2, 3, 4, 5, and 6 days, and the optical density (OD) at λ = 450 nm was monitored as a function of time. As illustrated in 1 significantly inhibited the proliferation of the WI26 cells at lower concentration, compared with both 2 and xiamenmycin. The extent of this inhibition of 1 increased with time, from a decrease of 13.8% at day one to a decrease of 38% at day six at a concentration of 15 μg/mL, compared with a control sample that was only treated with solvent  and could mimic the ppGpp binding effect on the mutated RNAP 26D +28.45° ; UV (MeOH) λmax = 206, 260 nm; CD  Δε201 +12.3, Δε202 +9.6, Δε205 +8.0, Δε207.4 +7.1, Δε213.6 +0.14, Δε217 −1.2, Δε245.2 +2.0, Δε259.8 +3.14, Δε283 +0.03; 1H and 13C NMR data, see m/z 290.1768 [M + H]+, , 288.1610 [M − H]−, .Compound 2: Yellow amorphous powder (MeOH); [α]30D +6.52° ; UV (MeOH) λmax 206, 260 nm; CD  Δε205.6 +6.7, Δε206.9 +5.5, Δε213 +20.1, Δε223.4 +0.85, Δε260 +9.23, Δε296.8 +1.0; 1H and 13C NMR data, see m/z 406.2210 [M + H]+, .Compound 1 and 2, as well as xiamenmycin on cell proliferation were determined using a standard Cell Counting Kit-8  assay according to the manufacturers instructions. WI26 cells at 70%–80% confluency were treated with a 0.25% trypsin and 0.02% EDTA solution, centrifuged and resuspended in DMEM supplemented with 10% FBS and antibiotics. The cells were then seeded in 96-well plates (100 μL/well) at an initial density of 2.5 × 104 cells/mL. The medium was replaced 24 h later by fresh DMEM with 10% FBS and antibiotics containing 15 μg/mL of compound 1, 30 μg/mL of compound 2, 30 μg/mL of xiamenmycin, or 0.1% DMSO . Subsequently, the medium was refreshed and the viabilities of the cells were measured by using a CCK-8 solution at day 0, 1, 2, 3, 4, 5, and 6, respectively. A proliferation measurement was performed by adding 10 μL of CCK-8 solution to each well and incubating the solutions at 37 °C for 1 h. The OD values of each well were measured at the primary wavelength λ = 450 nm using a Microplate Spectrophotometer . The data are shown as the means ± standard deviations (SD) of three independent experiments, each performed in triplicate.The effects of compounds t-test at significance levels of p < 0.05 to 0.001.Statistical differences were calculated using student’s paired Streptomyces xiamenensis, strain 318, and it has multiple anti-fibrotic effects. The deep sea-derived S. xiamenensis strain M1-94P was selected for further chemical and pharmaceutical studies of benzopyran compounds. The introduction of spontaneous rifampicin resistance combined with streptomycin resistance by introducing a mutated rpsL gene into M1-94P was performed to increase the production of its secondary metabolites. Two new benzopyran derivatives, xiamenmycin C (1) and D (2), were isolated from one of the Rif-Str double mutants, M6. The structures of 1 and 2 were identified through extensive spectroscopic data analysis. The isolation of the novel compounds increased the structural diversity of known benzopyran compounds, and the anti-proliferation bioactivities of 1 and 2 on human lung fibroblast behavior were investigated. Compared with xiamenmycin, the bioactivity of 1 is increased when the amino acid moiety was removed and compound 2 showed similar anti-fibrotic activity. Our work may be useful for a structure-activity relationship study of anti-fibrotic benzopyran compounds to develop leading drugs for medical treatment of excessive fibrotic disease from marine natural product.The benzopyran compound xiamenmycin can be obtained through the cultivation of a mangrove-derived strain of


Diabetes is a major risk factor for left ventricular dysfunction with cardiac and renal fibrosis. C-type natriuretic peptide (CNP) is a 22 amino-acid peptide produced mainly in the cardiac endothelium with potent cardiac unloading, anti-fibrotic and antihypertensive effects, but minimal renal actions. Using this knowledge we designed a natriuretic peptide CBA-NP by fusing a 6 AA sequence (KVLRRH) from BNP to the C-terminus and a 5 AA sequence (RMDRI) from ANP to the N-terminus of CNP to enhance beneficial renal effects while maintaining CNP’s inherent cardioprotective properties.Chronic treatment with CBA-NP will have direct anti-fibrotic and humoral effects in a rat model of diabetic cardiomyopathy.Using three groups of six male Wistar rats,  one dose of streptozotocin was administered to induce diabetes. One month after induction of diabetes ALZET pumps with 0.1μg/kg/min of CBA-NP or saline were serially implanted subcutaneously every 14 days over the course of 2 months. Cardiac function was assessed by echocardiography. Neurohormones by RIA. Fibrosis by picrosirius red staining. Ultrastructural features by electron microscopy.CBA-NP treatment attenuated LV hypertrophy (0.24±.01 mg/g body weight) compared to diabetic control (0.26±.01) and was comparable to the normal control (0.24±.01). LV interstitial and perivascular fibrosis percentage was significantly reduced in the CBA-NP treated group (3.27 to 1.80 and 3.87 to 1.77) as compared to the diabetic control. Ejection fraction (84.0± 1.2% vs. 78.0±1.7 %) and fractional shortening (48±1.2% vs. 41±1%) were significantly improved after CBA-NP treatment compared to diabetic control. Kidney cortical and medullary percent fibrosis was significantly reduced (4.58±0.80 to 2.16±0.14 and 4.43±0.6 to 1.23±0.3) after CBA-NP treatment as compared to the untreated group. GFR significantly improved (1.74±0.18 to 2.42±0.18) with reduction in glomerular basement membrane thickness. There was a significant decrease in plasma renin, aldosterone and BNP, while plasma cGMP increased in the treated group compared to the untreated group.CBA-NP treatment attenuated LV hypertrophy, reduced cardiac and renal fibrosis, and improved cardiac and renal function with suppression of renin and aldosterone, suggesting a potential therapeutic benefit in diabetic cardiomyopathy.


Phaleria macrocarpa (Scheff.) Boerl is frequently used in traditional medicine. The present investigation was undertaken with the purpose of developing pharmacopoeial standards for this species. Nutritional values such as ash, fiber, protein, fat, and carbohydrate contents were investigated, and phytochemical screenings with different reagents showed the presence of flavonoids, glycosides, saponin glycosides, phenolic compounds, steroids, tannins, and terpenoids. Our results also revealed that the water fraction had the highest antioxidant activity compared to the methanol extract and other fractions. The methanol and the fractionated extracts  of P. macrocarpa seeds were also investigated for their cytotoxic effects on selected human cancer cells lines  and a normal human fibroblast lung cell line (MRC-5). Information from this study can be applied for future pharmacological and therapeutic evaluations of the species, and may assist in the standardization for quality, purity, and sample identification. To the best of our knowledge, this is the first report on the phytochemical screening and cytotoxic effect of the crude and fractionated extracts of P. macrocarpa seeds on selected cells lines.In recent years, the utilization of certain medicinal plants as therapeutic agents has drastically increased.  Phaleria macrocarpa (Scheff.) Boerl is still relatively unknown in terms of its biochemical constituents and biological activity. P. macrocarpa is a plant commonly used in East Asian herbal medicines. P. macrocarpa is used as a remedy for a variety of ailments such as cancer, diabetes mellitus, allergies, liver and heart diseases, kidney failure, blood diseases, high blood pressure, and stroke. It is also used to treat various skin diseases including acne  × 100 (A is absorbance).Screenings of antioxidant activity of the crude methanol extract, chloroform, hexane, ethyl acetate, and water fractions of methods , 25. BasHuman cervical carcinoma cells (Ca Ski), hormone-dependent breast carcinoma cells (MCF-7), human breast adenocarcinoma cells (MDA-MB231), human ovarian carcinoma cells (SKOV-3), human colon carcinoma cells (HT-29), and noncancer human fibroblast cells (MRC-5) were purchased from the American Tissue Culture Collection .μg/mL penicillin or streptomycin , and 50 μg/mL kanamycin/amphotericin B . The cells were cultured in a CO2 incubator (5%) and kept at 37°C in a humidified atmosphere. The cultures were subcultured every 2-3 days and checked frequently under an inverted microscope  for any contamination.HT 29, Ca Ski, and MCF-7 cells were maintained in RPMI 1640 medium (Sigma), MDA-MB231 and SKOV-3 cells in Dulbecco's Modified Eagle's medium , and MRC-5 cells in Eagle's Minimum Essential medium , supplemented with 10% fetal bovine serum , 100 2 incubator at 37°C for 24 hrs. After reaching 70–80% confluence, each extract at concentrations of 1, 10, 25, 50, and 100 μg/mL (in 200 μL of 10% media) was added to the respective wells containing the cells. Wells with untreated cells were used as the negative control and cells exposed to doxorubicin were used as positive control. After 24, 48, and 72 hours, 10 μL MTT stock solution was added to each well. OD was then determined by measuring absorbance at 540 nm using an ELISA microplate reader. The percentage of inhibition (%) was calculated according to the following formula:50 value. The extract that gave IC50 of 30 μg/mL or less was considered active  assay was performed on the cultured cells in 96-well plates according to the method of Mosmann , 27. Brid active .μL with a split mode at a ratio of 1 : 20. The injection port temperature was set to 230°C and the detector/interface temperature was set to 250°C. The results were collected for 40 minutes. The total ion chromatogram obtained was autointegrated using ChemStation and the chemical compounds or components were analysed by comparison with the supplied mass spectral database .GC-MS was used to determine the molecular weight of the components collected and the purity of the collected extracts. Running conditions were as follows: oven temperature was programmed with an initial temperature of 100°C and increased at a ramp rate of 5°C/min and reached a final temperature of 300°C. The carrier gas or mobile phase used was helium and a flow rate of 1 mL/min was programmed. The mass spectrometry mode used was electron ionization (EI) mode with a current of 70 eV. The injection mode was programmed with a sample injection volume of 1 All data for each test are the average of triplicate experiments for comparison of values and were recorded as the mean ± standard deviation using Microsoft Excel software and statistical data analyses were performed using SPSS software. P. macrocarpa seeds is shown in  P. macrocarpa seeds was the hexane fraction (9.47%).The extracts were concentrated using a rotary evaporator  under reduced pressure at 35°C. The yield of extracts from P. macrocarpa seeds, a preliminary phytochemical investigation was carried out according to conventional methods. The results obtained from these experiments are summarized in In order to determine the types of phytoorganic constituents present in P. macrocarpa seeds. Small amounts of alkaloids, α-amino acids, cyanogenic glycosides, organic acids, reducing sugars, and starches were also found to be present.The phytochemical tests showed that there were secondary metabolites, including carbohydrate, flavonoids, glycosides, saponin glycosides, phenolic compounds, steroids, tannins, and terpenoids, present in different extracts of P. macrocarpa seeds may contribute to the presence of bioactivities such as antibacterial, an analgesic, an antifungal, an anti-inflammatory agent, and cytotoxicity. Moreover, the toxic chemical constituents, cyanogenic glycosides, were also present in the seeds.The main constituents such as flavonoids, glycosides, saponin glycosides, phenolic compounds, steroids, tannins, and terpenoids present inThe determination of nutritional values such as moisture, ash, fiber, protein, fat, and carbohydrate contents was carried out using the A.O.A.C method as well as the Lane and Eynon titration method and the results obtained are shown in It was found that the methanol extract had the highest flavonoid content (9.33 ± 0.8 mg/mL), followed by ethyl acetate, water, chloroform, and hexane fractions, which were 8.38 ± 1.0, 8.08 ± 0.3, 6.78 ± 1.1, and 4.18 ± 1.5 mg/mL, respectively. P. macrocarpa seeds exhibited the highest amount of total flavonol content, followed by water, ethyl acetate, chloroform, and hexane fractions, which were 8.93 ± 1.0, 6.13 ± 0.5, 4.29 ± 0.9, and 4.29 ± 0.7 mg/mL, respectively.The methanol extract (8.93 ± 1.1 mg/mL) of P. macrocarpa seeds extracts and fractions was expressed as gallic acid equivalents. The methanol extract of P. macrocarpa seeds exhibited the highest amount (7.20 ± 0.7 mg/mL) of total phenolics, followed by water, ethyl acetate, chloroform, and hexane fractions, which were 5.12 ± 1.4, 3.58 ± 1.1, 3.26 ± 1.0, and 2.63 ± 0.1 mg/mL, respectively.The total phenolic content of theThese data are shown in  P. macrocarpa seeds and to determine the antioxidant activity of its phytoconstituents. It is important to note that a lower IC50 value equals a higher scavenging activity. The scavenging activity was presented as the percentage of inhibition of DPPH free radicals  were investigated for cytotoxic effects in human cervical adenocarcinoma cells (Ca Ski), human hormone-dependent breast carcinoma (MCF-7), human colon adenocarcinoma cells (HT-29), human ovarian carcinoma cells (SKOV-3), human hormone-dependent breast carcinoma (MDA-MB231), and normal cells (MRC-5) using MTT cells proliferation assay at 24 hrs, 48 hrs, and 72 hrs, respectively. P. macrocarpa seeds showed excellent cytotoxic effects with IC50 value of 8.2 ± 4.66 μg/mL in Ca Ski cells at 24 hrs and 12.0 ± 2.2, 8.5 ± 1.68, and 3.0 ± 2.50 μg/mL in MCF-7 cells at 24 hrs, 48 hrs, and 72 hrs, respectively. The methanol extract also showed good cytotoxic activity in HT-29 cells with IC50 values of 29.3 ± 2.26, 25.0 ± 1.35, and 21.5 ± 3.30 μg/mL at 24 hrs, 48 hrs, and 72 hrs, respectively, in Ca Ski cells with IC50 values of 19.7 ± 0.92 μg/mL, and in SKOV-3 cells with IC50 values of 16.5 ± 2.52, 22.1 ± 2.47, and 36.0 ± 3.55 μg/mL at 24 hrs, 48 hrs, and 72 hrs, respectively. However, this extract had no cytotoxic effect in MDA-MB231 cells with IC50 >100 μg/mL, and the extract also had low cytotoxic effect in the MRC-5 cells with IC50 > 50 μg/mL.The methanol extract of P. macrocarpa seeds showed moderate cytotoxic effects with IC50 values of 45.2 ± 1.49 and 55.5 ± 1.97 μg/mL at 24 hrs and 48 hrs on MCF-7, 40.0 ± 3.15 μg/mL in HT-29 cells at 24 hrs, and 40.5 ± 3.52 and 50.0 ± 3.02 μg/mL in SKOV-3 at 24 hrs and 48 hrs. In addition, the hexane fraction also displayed low cytotoxic effect in MCF-7 at 72 hrs, HT-29 at 48 hrs and 72 hrs, and in SKOV-3 at 72 hrs. The IC50 value of treatment with hexane fraction in MCF-7 was 72.5 ± 1.52 μg/mL for 72 hrs exposure. IC50 values in HT-29 were 64.0 ± 2.03 and 75.0 ± 3.14 μg/mL at 48 hrs and 72 hrs, while IC50 value in SKOV-3 was 70.3 ± 3.53 μg/mL at 72 hrs. In contrast, the hexane fraction exhibited no cytotoxic effects in MDA-MB231 cells or MRC-5 cells.The hexane fraction of P. macrocarpa seeds exhibited the highest cytotoxic effect with IC50 value of 10.0 ± 1.31, 8.2 ± 1.04, and 22.0 ± 1.86 μg/mL at 24 hrs, 48 hrs, and 72 hrs in Ca Ski cells, 9.5 ± 2.95, 8.7 ± 1.59, and 21.0 ± 1.98 μg/mL at 24 hrs, 48 hrs, and 72 hrs on HT29, and 24.8 ± 2.06, 16.5 ± 3.21, and 9.00 ± 2.6 μg/mL in SKOV-3 cells. The chloroform fraction exhibited moderate cytotoxic effect in MCF-7 with IC50 value of 57.5 ± 2.64, 40.0 ± 1.48, and 46.5 ± 3.45 μg/mL for 24 hrs, 48 hrs, and 72 hrs, respectively. In contrast, the chloroform fraction had no cytotoxic effect against the MDA-MB231 cells and normal MRC-5 cells with IC50>100 μg/mL.The chloroform fraction of P. macrocarpa seeds exhibited the highest cytotoxic effect with IC50 < 25 μg/mL in SKOV-3 cells, MDA-MB 231 cells, MCF-7 cells, and Ca Ski cells. On the other hand, the ethyl acetate fraction exhibited low cytotoxic effect in MRC-5 normal cells with IC50 value of 35 μg/mL. The ethyl acetate fraction exhibited the highest cytotoxic effect in all selected cells . The water extract showed no cytotoxic effect against all selected cancer cell lines with IC50>100 μg/mL and exhibited a mild cytotoxic effects against MRC-5 cells. The corresponding data is shown in The ethyl acetate fraction of P. macrocarpa seeds using GC-MS. They were methyl stearate, oleic acid, methyl oleate, linoleic acid, methyl linolenate, and palmitic acid. Moreover, a GC/MS analysis of the chloroform fraction of P. macrocarpa seeds showed the presence of methyl myristate, palmitic acid, methyl oleate, methyl linoleate, oleic acid, and (z)- and 9,17-octadecadienal, (z). A comparison with the NIST mass spectral library  and Adams (2001) confirmed the identity of the compounds. The chemical structures of the identified compounds in the hexane and chloroform fractions of P. macrocarpa seeds are as shown in Six compounds were identified from the hexane fraction ofA longer shelf life can be achieved by reducing moisture content. Thus, moisture content is a critical factor for the stability of an extract. Moisture enhances fungal and bacterial growth, therefore decreasing the longevity of the extract. The time it takes for plant material to deteriorate depends on how much water the plant material contains.β-glucans, gums, and pectin and hydrocolloids. These components can behave as proantioxidants [ P. macrocarpa seeds were mainly made up of carbohydrates, followed by crude fiber, proteins, fat, moisture, and ash. High-protein and low-carbohydrate diets are often effective for weight loss. Dietary fiber can aid in digestion, is helpful for weight management, and reduces constipation. However, many high-protein and low-carbohydrate foods are low in fiber. Women aged 50 and older need at least 21 grams of fiber daily, whilst those aged 19 to 50 require 25 grams, men aged 50 and older need 30 grams, and those between the age of 19 to 50 should consume at least 38 grams of fiber.Carbohydrates play a vital role in the immune system, fertilization, pathogenesis, blood clotting, and human development. Foods that contain carbohydrates can raise blood glucose and the three main types of carbohydrate are starches, sugars, and fiber. A macronutrient protein consists of amino acids which is required for proper growth and human body function. The seeds were found to contain a high level of crude fiber which is a potential source of phenolic antioxidants. The major components of fibers are cellulose, hemicelluloses, lignin, oxidants , 29. In  P. macrocarpa seeds to be used in the treatment of diseases such as diabetes, diarrhea, and dysentery. Our preliminary phytochemical studies revealed that the chemical components of P. macrocarpa seeds also include flavonoids, phenolics, steroids, tannins, terpenoids, glucosides, saponins, and carbohydrates.Steroids possess biological activities which are insecticidal, cardiotonic, and antimicrobial activities and these are potentially useful for development into therapeutic drugs. Tannins are known to be important for their antiviral, antibacterial, antiparasitic effects, anti-inflammatory, antiulcer. and antioxidant properties –33. Sapoβ-D-glucoside, dodecanoic acid, palmitic acid, ethyl stearate, sucrose [β-D-glucoside) [β-D-glucoside [ P. macrocarpa. The hexane extract of the seeds was found to contain methyl myristate, methyl stearate, oleic acid, methyl oleate, linoleic acid, methyl linoleate, palmitic acid, methyl palmitate, 6-octadecenoic acid, and 9,17-octadecadial, β-sitosterol as major components. A further 30% to 40% of components were not identified as these were polar compounds that require HPLC analysis. Other compounds reported in the literature for the seeds, leaves, and fruits were not identified in this investigation. It is probable that the components reported in the literature may be present in other fractions .Previous researchers reported that kaempferol, myricetin, naringin, quercetin, rutin , 29-norc sucrose , pinores sucrose , 39, sap sucrose –42, icarucoside) , and 2,4lucoside  were iso50 values of free radical and reducing power in methanol with HCl of P. macrocarpa seeds were 245.0 ± 1.94 and 150.2 ± 1.28 [50 value followed by methanol, ethyl acetate, hexane, and chloroform fraction.Phenolics are able to transfer protons to radicals . DPPH, w2 ± 1.28 . In our  P. macrocarpa seeds may be responsible for the traditional use of the plants in treating cancer, inflammations, and allergies. Recently, it has been shown that phenolic compounds in crude methanol and ethyl acetate extract of P. macrocarpa leaves displayed good antioxidant and antimicrobial activites [ P. macrocarpa seeds [The presence of flavonoids inctivites . Hendra pa seeds . In thispa seeds  and phen P. macrocarpa seeds and fruit meat were not toxic to normal human cells, but slightly toxic to a Vero cell line [ P. macrocarpa seed fruit also showed toxicity towards T47D breast cancer cell line through COX-2 expression inhibition [ P. macrocarpa seeds and this compound displayed excellent cytotoxicity in brine shrimp [ P. macrocarpa seeds exhibited mild cytotoxic effect against HepG2 cells  [ P. macrocarpa seeds was found to pose cytotoxic effect against HT-29, MCF-7, Cas Ki, and SKOV-3 cell lines  and mild toxicity on normal cell lines. There were significant cytotoxic effects on selected cancer cells lines that were both time- and dose-dependent manner due to the presence of many secondary metabolites.MTT assay is a sensitive and reliable colorimetric assay that uses quantitative measurements to calculate the viability, proliferation, and activation of cells. This method is commonly applied to screen anticancer agents . The mosell line . The ethe shrimp . The eth0 μg/mL) . Previou0 μg/mL) . In our The phytochemical screening also indicated the presence of a small amount of cyanogenic glycosides. Cyanogenic glucosides which can cause acute cyanide poisoning cause rapid respiration and pulse, decrease the blood pressure, and induce vomiting, diarrhea, headache, dizziness, and so on. Thus, these components have to be removed  prior to consumption for therapeutic purposes.P. macrocarpa seeds exhibited antioxidant and cytotoxic activities. It is highly probable that these activities are due to the presence of phenolic and flavonoid compounds in appreciable amounts in the plant. Furthermore, the cytotoxicity activity suggested that the seed may contain a potential anticancer agent. The outcome of this study is encouraging, demonstrating the potential for the P. macrocarpa as a source of multiple therapeutic agents.


P < .001) and systolic (P < .05) and diastolic (P = .03) blood pressure on the second day were significantly lower in the group inhaling eucalyptus than that inhaling almond oil. Heart rate, CRP, and WBC, however, did not differ significantly in the two groups. In conclusion, inhalation of eucalyptus oil was effective in decreasing patient's pain and blood pressure following TKR, suggesting that eucalyptus oil inhalation may be a nursing intervention for the relief of pain after TKR.Eucalyptus oil has been reported effective in reducing pain, swelling, and inflammation. This study aimed to investigate the effects of eucalyptus oil inhalation on pain and inflammatory responses after total knee replacement (TKR) surgery. Participants were randomized 1 : 1 to intervention group  or control group . Patients inhaled eucalyptus or almond oil for 30 min of continuous passive motion (CPM) on 3 consecutive days. Pain on a visual analog scale (VAS), blood pressure, heart rate, C-reactive protein (CRP) concentration, and white blood cell (WBC) count were measured before and after inhalation. Pain VAS on all three days ( Eucalyptus globulus) oil has been widely used as a folk medicine to treat upper respiratory infections, gastritis, and diabetes [Osteoarthritis, the most prevalent musculoskeletal disorder throughout the world, is a common chronic disease that causes pain, restricts activity, and reduces quality of life . Osteoardiabetes .α-pinene and 1,8-cineole and acts as an antioxidant, with strong radical scavenging activity [Eucalyptus oil contains activity . In a moactivity . Electroactivity . Moreoveactivity .Although eucalyptus oil has been found to reduce pain and suppress edema and inflammation in animal models, its effects on pain in patients who have undergone TKR have not been determined. We therefore assessed whether eucalyptus oil inhalation could effectively reduce pain and inflammatory responses in patients who have undergone TKR.Patients were randomly assigned using table of random numbers to inhalation of eucalyptus oil or almond oil  during continuous passive motion (CPM) after TKR, with both investigators and subjects blinded to treatment assignment. Based on an effect size of 0.80, a statistical power of 0.80, and a significance level of 0.05, the minimum number of patients required to compare differences between the experimental and control group was estimated to be 26 patients per group. Twenty-eight subjects were originally assigned to each group, with 52 completing the study, 25 in the experimental and 27 in the control group.Patients diagnosed with osteoarthritis by the same physician and who underwent TKR were invited to participate. All subjects (1) had been prescribed pain medications, including oxycodone hydrochloride, fentanyl, nonsteroidal anti-inflammatory drugs (NASID), and antibiotic pills, (2) had no complications or other inflammatory diseases after surgery, (3) were not being treated with any antidepressant, hormone, or aroma therapy, (4) did not smoke, (5) were conscious and oriented, and (6) had a pain score on a visual analog scale (VAS) >4 before aroma inhalation. The study design and protocol were approved by the Ethical Review Committee of the Korea University Medical Center (Code: ED11285), and all participants provided written informed consent.μm film thickness, Agilent, USA). Carrier gas was helium and flowrate was 1.0 mL/min. The injector temperature was increased to 280°C. And samples (1.0 μL) were injected at a split ratio of 50 : 1. The column temperature was initially maintained at 50°C (hold for 3 min), ramp to 240°C at 3°C/min, and finally held for 10 min. The ion source and transfer line temperature were set at 230°C and 250°C. The mass spectrometer was operated in the electron impact ionization mode (70 eV).Gas chromatography-mass spectrometry (GC-MS) was performed to analyze compositions of eucalyptus oil. GC-MS analysis was performed using an Agilent 7890 gas chromatograph with a 5975 inert mass spectrometer (USA), and analytical capillary column was performed on HP-Innowax . Eucalyptus oil was dissolved at a concentration of 3% (v/v) in almond oil. In experimental group, eucalyptus oil  was placed onto a 4 × 2 inch gauze pad, which was positioned between the nose and philtrum for 30 minutes of CPM on 3 consecutive days, beginning on the third day after surgery. The control group received the same treatment of experimental group except for eucalyptus. Eucalyptus oil and almond oil have a similar color and were packed same-shaped bottles. All experiments were carried out separately. The compounder was the only one who knows which participant affiliated to which group according to the assigned number on bottle. Patients and investigator were not informed about the types and effects of aroma oil.Pain was measured using the VAS pain score. It is also used in most studies of anxiety, consists of a horizontal scale, ranging from 0 (no pain) on the left side and 10 (extreme pain) on the right side . PatientIn this study, CRP was measured by a Latex Immunoassay with LX-2200  and WBC was measured by semiconductor flow cytometry with an XE-5000 .Blood samples were collected before breakfast on the first day of aroma inhalation and on days 4 and 7 for measurements of C-reactive protein (CRP) concentrations and white blood cell (WBC) count. Pain VAS, blood pressure, and heart rate were measured before and after each 30 min inhalation session.t-tests, Fisher's exact tests, and Chi-square tests, as appropriate. All data are presented as frequency, percentages, and standard error of the mean (SEM) or standard deviation (SD). Differences in the VAS pain scores before and after aroma inhalation within each group were compared using the Wilcoxon's rank-sum test and paired t-test, and differences between groups were analyzed using Mann-Whitney U-tests and unpaired t-tests.All statistical analyses were performed using the Statistical Package for the Social Sciences, SPSS 20.0. The demographic and clinical characteristics of the two groups were compared using ρ-cymene (8.55%), γ-terpinene (5.87%), and α-pinene (4.95%).A total of 31 compounds were identified . The maj2, and all 52 were overweight/obese (BMI ≥ 25 kg/m2). Thirty-seven patients (71.2%) underwent a unilateral TKR and 15 (28.9%) underwent a simultaneous, bilateral TKR. The mean durations of osteoarthritis in the eucalyptus and almond oil groups were 8.5 ± 5.7 years and 6.0 ± 5.2 years, respectively. Before aroma inhalation, VAS pain score, systolic blood pressure, diastolic blood pressure, heart rate, CRP, and WBC count were similar in the two groups  group. Demographic and disease-associated characteristics were similar in the two groups, including type of surgery, duration of osteoarthritis, and treatments with oral antihypertensive and antidiabetic agents . The meao groups .P < .001, VAS pain scores after aromatherapy on days 1–3 decreased 1.1 ± 0.2, 1.2 ± 0.2, and 1.2 ± 0.2 points, respectively, from the scores before inhalation . In the Relative to pretreatment heart rate, heart rate in the eucalyptus oil group increased 0.3 ± 1.6 beats/min on day 1 of CPM and decreased 1.7 ± 1.7 beats/min and 0.6 ± 1.0 beats/min on days 2 and 3, respectively . The heaP < .05, P = .03, Systolic blood pressure on days 1–3 decreased 0.8 ± 1.9 mmHg, 4.8 ± 2.2 mmHg, and 2.0 ± 2.0 mmHg, respectively, in the eucalyptus oil group, while increasing 0.4 ± 1.7 mmHg, 3.3 ± 2.2  mmHg, and 1.9 ± 1.6 mmHg, respectively, in the control group  . On daySerum CRP concentrations before inhalation and on days 4 and 7 were 7.2 ± 3.9, 53.5 ± 6.8, and 48.8 ± 11.0 mg/L, respectively, in the eucalyptus oil group, and 4.89 ± 2.0, 68.2 ± 8.2, and 46.8 ± 7.7 mg/L, respectively, in the control group . Althoug3/μL, 7,062.0 ± 377.9 × 103/μL, and 7,450.0 ± 383.5 × 103/μL, respectively, in the eucalyptus oil group, and 6,793.3 ± 268.8 × 103/μL, 7, 112.6 ± 336.9 × 103/μL, and 7,970 ± 502.4 × 103/μL, respectively, in the control group (WBC counts before inhalation and on days 4 and 7 were 6, 513.2 ± 417.0 × 10ol group . None ofSince eucalyptus oil has been reported effective in reducing pain and suppressing inflammation in the various animal models, we tested whether inhalation of eucalyptus oil affected pain, blood pressure, heart rate, CRP concentration, and WBC count following TKR in patients with osteoarthritis.Pain is an emotion which is quintessentially subjective and personal , 14. To Hypericum perforatum is considered to inhibit the synaptosomal uptake of serotonin [Serotonin has been considered to have an important role in the control of pain . Activaterotonin . Also, lerotonin . Therefoerotonin , 20. Recerotonin . Therefoerotonin , 23. Otherotonin . Thus, tEucalyptus oil has been used to treat influenza infection, owing to its anti-inflammatory and antibacterial effects . MoreoveIn summary, this study, which investigated the effects of eucalyptus oil inhalation on patients who underwent TKR, showed that eucalyptus oil inhalation was effective in reducing patient's subjective pain and blood pressure after surgery. These findings suggest that the inhalation of eucalyptus oil might be a valuable nursing intervention for pain relief after TKR.


Unphysiological lung strain  may cause ventilator-induced lung injury (VILI) . WhetherIn 20 healthy, mechanically ventilated pigs, FRC and lung weight were measured by computed tomography. Animals were then ventilated for up to 54 hours with a TV set to produce a predetermined strain. At the end, lung weight was measured with a balance. VILI was defined as final lung weight exceeding the initial one.n = 9) or markedly augmented . In the two groups, strain was 1.38 ± 0.68 and 2.16 ± 0.50 (P < 0.01), respectively. VILI occurred only when lung strain reached or exceeded a critical threshold, between 1.5 and 2.1 (Figure Lung weight either did not increase at all (no-VILI group; lung weight change -73 ± 42 g, In animals with healthy lungs VILI only occurs when lung strain exceeds a critical threshold.


Surgical ventricular restoration (SVR) after anterior myocardial infarction with post infarction aneurysm, significantly improves left ventricular (LV) performance. To describe mid-term postoperative changes of shape and performance of the newly created LV and mitral valve function through the classical (SI) and apical (ASI) left ventricular sphericity index, and determinants of mitral regurgitation.A total of 26 consecutive patients underwent SVR between November 2002 and January 2004 at our institution. Transthoracic echocardiogram (TTE) was performed before, 3.1 months).±immediately postoperatively and during follow up . EDV, ESV and EF improved , without significant change in E/A and DT  but with improvement in IMP±69,99 to 209,64±0,74 NS; 207,54±1,29 0.047).There was a statistically significant<0,15 p±0,21 to 0,501± and ASI  as in the decrease in degree of MR severity<0,13 p±0,10 to 0,67±0,76 0.018).SI<0,49 p±1,13 to 0,17±immediately postoperatively (0,76 and ASI enable simple and clinically useful definition of complex morphological and functional changes of the distal part of the left ventricle, as preoperatively, and postoperatively, after the SVR and formation of the new LV apex.


Introduction. Lymphocytic infiltration and specific lymphocytes subsets may play important roles in papillary thyroid carcinoma (PTC) progression and prognosis. In this study, we try to understand the influence of 131I radioablation on the important lymphocytes subtypes of regulatory T and B cells (Tregs and Bregs). Methods. Peripheral blood mononuclear cells from 30 PTC patients before and after 131I therapy, and 20 healthy donors were collected. The expression of Tregs (CD4+CD25+CD127−/low) and B cell (CD5+CD19+) and production and secretion of interleukin 10 (IL-10) were analyzed by FACS and ELISA assay, respectively. Results. For Tregs percentage in peripheral blood lymphocytes, there was no difference between pretreatment and control and between posttreatment and control. Compared with pretherapy, increased Tregs infiltration was noted in posttherapy (P < 0.05). Although no difference was between pretreatment and control, compared with these two groups, decreased CD19+ and CD5+CD19+ B cell percentage in posttreatment was observed (P < 0.05). Among these groups, no significant difference was displayed in intracellular IL-10 production and extracellular IL-10 secretion. Conclusions. 131I Radioablation increased Tregs and decreased CD19+ and CD5+CD19+ B cells percentage after treatment. However, it has no effect on IL-10 and lymphocytes in peripheral blood. Therefore, longer follow-up of Tregs and Bregs should be further investigated. Thyroid cancer is the most common endocrine malignancy, which constitutes approximately 1% of all human malignancies . Papilla+ T cells, which play an important role in the immune response  × 109/L) is within normal levels (1.1–3.2 × 109/L). However, there is significant difference between the pretreatment and posttreatment group (P < 0.05).The absolute number of lymphocytes in the pretreatment ([2.45 ± 0.68] × 10+ T cells and CD4+CD25+CD127−/low T cells (Tregs), respectively %, (30.38 ± 8.00)%, and (34.74 ± 8.84)%, respectively. There was no significant difference among the above groups (P > 0.05)  . + T cells) in blood of PTC patients before 131I therapy (2.52 ± 0.87)%, Tregs was significantly higher in the posttherapy group (3.23 ± 0.84)% (P < 0.05)   .+ and CD5+CD19+ B cells, respectively %, (3.99 ± 1.16)%, and (6.96 ± 1.24)%, respectively. Blood samples of patients with PTC and the healthy donors were analyzed by flow cytometry for CD19ectively . The CD1P > 0.05), but the significant difference was noted between posttreatment and control (P < 0.05)  in blood of PTC patients before the 131I ablation (2.07 ± 0.97)%, CD5+CD19+ B cells were significantly lower in the posttreatment group (1.63 ± 0.59)% (P < 0.05). CD5+CD19+ B cells in the healthy donors were (2.39 ± 0.43)%. Similar to the results of CD19+ B cells, there was no significant difference between pretreatment and control (P > 0.05), but the significant difference was observed between posttreatment and control, respectively (P < 0.05)  . Compare Figures .+CD19+ B cell percentage by flow cytometry analysis in the pretreatment, posttreatment, and the healthy donors was (6.77 ± 5.80)%, (11.96 ± 17.25)%, and (11.72 ± 10.81)%, respectively. There was no significant difference among these groups (P > 0.05) (The intracellular production of IL-10 in CD5 > 0.05) . + B cells by ELISA analysis in the pretreatment, posttreatment, and the healthy donors was 9.02 ± 1.55, 9.44 ± 1.18, and 9.79 ± 0.57 pg/mL, respectively. There was no significant difference among these groups (P > 0.05) (The IL-10 production by CD5 > 0.05) . + B cells (putative B cells that have regulatory functions) are studies in blood samples of PTC patient before and after 131I ablation. To our knowledge, no previous study focused on this control research.The lymphocytic infiltration is frequently observed in PTC, and specific lymphocytes subsets may be the important regulators of PTC progression and prognosis , 27, 28.131I treatment were significantly higher. However, the posttreatment Tregs percentage in peripheral blood lymphocytes was found to have no significant difference with the healthy donors. The main function of human immune system is the body protection from a diverse range of agents, including tumor cells and radionuclide [131I, which was not beyond the self-tolerant extent and normalized the numbers of CD4+ T cells. Moreover, it is demonstrated that 131I ablation for PTC patients was a safe and effective therapy. Although Tregs have been studied and analyzed in many tumors, such as breast cancer, pancreas cancer, and melanoma –19, the + T cell activation and are involved in immune pathology through the production of cytokines that regulate T-cell function [+ B cells and between the pretreatment and control of CD5+CD19+ B cells. However, compared with healthy donors and PTC patients before 131I ablation, both CD19+ and CD5+CD19+ B cells after the 131I therapy were significantly decreasing. The reasons are probably as followed: compared with T cells, B cells are more sensitive to radiation and prone to radiation-induced inhibition [131I radioablation, regulatory B cells prior to regulatory T cells play more important role in the regulation of peripheral blood lymphocytes.In addition to Tregs, certain B cell subpopulations could also exhibit potential regulation of immune response by functioning as cellular adjuvants for CD4function –33. Givefunction –23. In thibition . Moreovehibition , 36. Our+ T cells and Tregs. However, the numbers of posttreatment CD19+ and CD5+CD19+ B cells were lower than those of pretreatment, which suggests that the synthesis and secretion of IL-10 were relatively increased. It could develop and maintain that posttherapy Tregs were higher than pretherapy ones in this study.IL-10 was initially associated with Th2 cells and was described to inhibit Th1 cytokine production –39. Howe131I, only one time-point after treatment was chosen, which couldn't reflect the dynamic change in patients' peripheral blood lymphocytes. Another limitation is the small population. Large sample of analysis of regulatory T and B cells in patients with thyroid papillary carcinoma before and after 131I radioablation should be further investigated. Moreover, there were some potential limitations in this study. One is considering the clinical effect of 131I radioablation increased Tregs percentage and decreased CD19+ and CD5+CD19+ B cells percentage at one month after treatment. However, it has no effect on the production and secretion of IL-10, and the percentage and absolute number of lymphocytes in peripheral blood are within normal levels. Therefore, longer follow-up of changes of regulatory T and B cells should be further investigated.