Organic compounds in re-circulated leachates of aerobic biological treated municipal solid waste

Biodegradation of organic matter is required to reduce the potential of municipal solid waste for producing gaseous emissions and leaching contaminants. Therefore, we studied leachates of an aerobic-treated waste from municipal solids and a sewage sludge mixture that were re-circulated to decrease the concentration of biodegradable organic matter in laboratory-scale reactors. After 12 months, the total organic C and biological and chemical oxygen demands were reduced, indicating the biodegradation of organic compounds in the leachates. Curie-point pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and pyrolysis-field ionization mass spectrometry (Py-FIMS) revealed that phenols, alkylaromatic compounds, N-containing compounds and carbohydrates were the predominate compounds in the leachates and solid waste. Leachate re-circulation led to a higher thermal stability of the residual organic matter as indicated by temperature-resolved Py-FIMS. Admixture of sewage sludge to solid waste was less effective in removing organic compounds from the leachates. It resulted in drastic higher and more bio-resistant loads of organic matter in the leachates and revealed increased proportions of alkylaromatic compounds. The biodegradation of organic matter in leachates, re-circulated through municipal solid waste, offers the potential for improved aerobic waste treatments and should be investigated on a larger scale.     

Impacts of long-term application of organic fertilizers on soil quality parameters in reclaimed loess soils of the Rhineland lignite mining area

On the basis of long-term field experiments, the impact is demonstrated of the periodic application of organic fertilizers on the accumulation of organic matter and the development of the micro-pollutant content of reclaimed loess soils of the Rhineland lignite mining area under agricultural use. The oldest of these experiments (‘Berrenrath Humus Accumulation Experiment’) was begun in 1969. The results show that the regular input of organic matter (e.g. manure, waste compost, sewage sludge) favors the accumulation of soil organic matter (SOM). However, the type of organic material applied seems to be less important to the long-term accumulation process than the application rate. This is also true for composted and uncomposted manure, if the decay of organic matter during the composting process is taken into account. Nevertheless, the application of similar amounts of organic C in the form of manure resulted in a higher accumulation of SOM in a nitrogen-reduced farming system. Depending on the treatment, accumulation rates were between 0.02 and 0.08% SOM per year with values decreasing with time. From these results, it is estimated that reclaimed soils will take much longer to reach the former SOM level than was previously assumed. However, it is important to determine which SOM level is adequate for different soil functions (e.g. production function, filter and buffer function, transformation function), and whether the young SOM of reclaimed soils has the same properties as older SOM in undisturbed topsoils. As was expected, long-term fertilization with sewage sludge and waste compost led to an accumulation of some micro-pollutants in the topsoils treated. Nevertheless, the observed concentrations are quite low compared to background levels in topsoils of rural regions in North Rhine-Westphalia. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of t-butyldimethylsilylation in the gas chromatographic/mass spectrometric analysis of physiologic compounds found in plasma using electron-impact ionization

The use of N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide to prepare the t-butyldimethylsilyl derivatives of a number of organic compounds (selected amino acids, alpha-keto acids, ketone bodies, free fatty acids, urea, glycerol, lactate, and pyruvate) is reported. These derivatives are particularly useful for gas chromatographic/mass spectrometric analysis involving the use of stable isotopes and selected ion monitoring, since a peak of sufficient abundance at 57 mass/charge units below the molecular ion was always present, and was the result of the loss of one t-butyl group. In each case, this fragment contained the entire skeleton of the original compound, which permitted easy analysis using electron-impact ionization of these compounds alone or when labeled with stable isotopes in any nonexchangeable position.     

草原区矿产开发对景观格局和初级生产力的影响——以黑岱沟露天煤矿为例

煤炭是我国的主要能源,大型露天煤矿的开发推动了地区经济与社会的发展,但同时也引发了区域生态环境问题。因此,探讨矿产开发对区域景观格局的影响,并阐明景观格局动态与生态系统初级生产力的关系,对生态环境的保护具有重要意义。以内蒙古草原区的黑岱沟露天煤矿为例,利用3S(RS、GIS和GPS)技术,在野外实地调查的基础上,分析了1987年以来矿产开发导致的土地利用/覆盖变化、景观格局动态及其与生态系统初级生产力之间的关系,主要结果如下:(1)提出了界定最适研究区范围的方法,认为沿矿区边界向外建立10 km的缓冲区是该研究最适研究区域的大小;(2)草地和耕地是研究区主要的土地利用/覆盖类型,但是在过去20多年间其面积在逐渐减少,而工矿仓储用地及住宅用地面积在急剧增加;(3)矿产开发导致景观格局发生变化,并且在两种不同的空间尺度(研究区和矿区)上表现出总体变化趋势的一致性,但在后期有较大差异;(4)初级生产力变化呈现下降趋势,并且矿区的降低更为突出;(5)以生长季降水量为控制因子的偏相关分析表明,景观配置(平均斑块周长面积比、景观形状指数)与初级生产力呈正相关关系,在研究区尺度上尤其显著,但在矿区尺度上,景观结构组成(斑块密度、均匀度指数和多样性指数)更为重要,与初级生产力呈显著负相关;(6)受复垦规模和演替进程的影响,局限在矿区排土场上的植被重建尚不能改变研究区尺度景观-生态系统初级生产力之间的关系。可见,景观格局的变化以及景观格局与生态系统初级生产力之间的关系均存在尺度依赖性。     

Generalized N-Acetylneuraminic Acid Storage Disease: Quantitation and Identification of the Monosaccharide Accumulating in Brain and Other Tissues

Abstract: Brain and other tissues from a patient with extensive neonatal as-cites and clinical symptoms suggestive of a severe neurovisceral storage disorder were examined following autopsy for the accumulation of oligosaccharides. This carbohydrate analysis revealed the presence of large amounts (3–21 μmol/g fresh weight) of sialic acid in brain, liver, and kidney tissue as the major abnormality. Exhaustive characterization of the accumulating material by gel filtration, gas-liquid chromatography, thin-layer chromatography, and GLC-mass spectrometry positively identified the saccharide as free N -acetylneuraminic acid. Based on the accumulation of only free N -acetylneuraminic acid in the tissue of this patient, and normal activities of lysosomal enzymes involved in the catabolism of cellular glycoproteins, this storage disorder appears to result from a previously unreported defect in glycoconjugate metabolism.     

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti‐aging and metabolic effects of sirtuins

Sirtuins are a family of NAD⁺‐dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti‐aging effects of human homologues of sirtuins, SIRT1‐7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti‐aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI‐TOF/TOF‐MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co‐immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti‐aging and metabolic regulatory activities of sirtuins.     

黔中煤矿区矸石堆场周边土壤藻类群落变化及其影响因素

通过对贵阳市花溪区麦坪乡煤矿区矸石堆场周边土壤进行采样分析,研究了煤矸石影响下土壤藻类群落组成和生物量的变化以及生物量、物种多样性指数与土壤因子之间的相关关系。结果表明:该区自然土壤藻类共计5门43属,其中蓝藻门(Cyanophyta)13个属、绿藻门(Chlorophyta)17个属、硅藻门(Bacillariophyta)7个属。受煤矸石污染后,土壤藻类的种类和数量明显减少,污染严重区域土壤藻类中蓝藻门仅有3个属、绿藻门4个属;香浓指数与有效铁、有效锰及速效磷含量之间存在极显著的相关性;物种丰富度与pH值和速效磷含量呈显著相关。污染严重区土壤藻类叶绿素a含量比对照区减少了93.1%;藻类生物量与pH值呈显著正相关,与有效铁含量呈显著负相关。调查区内土壤pH值和有效铁含量是影响土壤藻类生长的重要因子。     

One-step Metabolomics: Carbohydrates,Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.     

High-performance liquid chromatography-coordination ion spray-mass spectrometry (HPLC-CIS/MS): a new tool for the analysis of non-polar compound classes in plant extracts using the example of Piper methysticum Forst

An efficient method to characterise complex plant extracts is described using the example of Piper methysticum Forst. (kava; Piperaceae). The method is based on the on-line coupling of high-performance liquid chromatography to a new detection technique: coordination ion spray-mass spectrometry (CIS/MS). CIS/MS is a universal, novel ionisation technique improving selectivity as well as sensitivity. Charged complexes were formed through addition of central complexing ions such as sodium, silver and cobalt. The advantages of CIS/MS detection compared with the electrospray ionisation detection are discussed. The experimental set-up and the application of this simple and robust technique is described to show the its various fields of application in the analysis of plant extracts.     

Giardia duodenalis: direct experimental evidence for the absence of a glycosylphosphatidylinositol anchor in a variant surface protein

The trophozoites of Giardia duodenalis express variant surface proteins (VSPs) that cover the entire surface of the cell and can be altered by antigenic variation. In the present study, a VSP (VSPH7) expressed by the Giardia GS isolate was purified using Triton-X-114 extraction/phase partitioning and a combination of column chromatography methods. The purified VSP was typed by mass spectrometric fingerprint mapping and peptide sequencing and found to share 58-99.8% peptide identity with the VSPH7 protein sequence previously deduced from the cloned cDNA. Carbohydrate compositional analyses consistently showed the presence of galactose in the VSP preparations but a direct association of carbohydrate with the VSPH7 could not be established. Analysis of the C-terminal part of the purified VSPH7 by off-blot myo-inositol analysis provided for the first time direct experimental evidence that this protein is not modified via a GPI lipid.     

Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: Total and free testosterone reference ranges

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 μL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y = 1.02x + 1.5, r² = 0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low-testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.     

小地老虎中国种群性信息素的成分鉴定及田间试验（英文）

研究了小地老虎中国种群的性信息素组分,3日龄处女蛾单腺体性信息素提取物中性信息素的含量非常低。GC和GC-MS分析表明,小地老虎性信息素含有5种成分：顺-7-12碳乙酸酯（A）、顺-9-14碳乙酸酯（B）、顺-11-16碳乙酸酯（C）、顺-5-10碳乙酸酯（D）和顺-8-12碳乙酸酯（E）。它们的含量分别为：（0.245±0.098）、（0.080±0.031）、（0.089±0.03）、(0.085±0.031)和 (0.105±0.065)ng, 这5种物质的百分比分别为40.451±13.66、13.176±5.279、14.943±5.142、14.392±6.10和17.225±9.792,前3种物质的百分比为58.75±9.429、18.91±7.539和22.34±7.209。田间试验表明,性信息素单一组分均未诱到雄蛾,AB以3∶1的比例配成的诱芯对雄蛾有一定的引诱活性,一个诱捕器平均诱捕到2.6头。ABC组分以3∶1∶1的比例配成的诱芯对雄蛾引诱活性显著增强,一个诱捕器平均诱捕量为7.40头,是AB（3∶1）诱芯的2.8倍。诱芯中性信息素的含量对诱蛾活性有明显的影响,在剂量为200 µg时的平均诱捕量最高。     

Leucine-nitrogen metabolism in the brain of conscious rats: its role as a nitrogen carrier in glutamate synthesis in glial and neuronal metabolic compartments

The source of nitrogen (N) for the de novo synthesis of brain glutamate, glutamine and GABA remains controversial. Because leucine is readily transported into the brain and the brain contains high activities of branched-chain aminotransferase (BCAT), we hypothesized that leucine is the predominant N-precursor for brain glutamate synthesis. Conscious and unstressed rats administered with [U-13C] and/or [15N]leucine as additions to the diet were killed at 0-9 h of continuous feeding. Plasma and brain leucine equilibrated rapidly and the brain leucine-N turnover was more than 100%/min. The isotopic dilution of [U-13C]leucine (brain/plasma ratio 0.61 +/- 0.06) and [15N]leucine (0.23 +/- 0.06) differed markedly, suggesting that 15% of cerebral leucine-N turnover derived from proteolysis and 62% from leucine synthesis via reverse transamination. The rate of glutamate synthesis from leucine was 5 micro mol/g/h and at least 50% of glutamate-N originally derived from leucine. The enrichment of [5-15N]glutamine was higher than [15N]ammonia in the brain, indicating glial ammonia generation from leucine via glutamate. The enrichment of [15N]GABA, [15N]aspartate, [15N]glutamate greater than [2-15N]glutamine suggests direct incorporation of leucine-N into both glial and neuronal glutamate. These findings provide a new insight for the role of leucine as N-carrier from the plasma pool and within the cerebral compartments.     

Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays

The feasibility of multi-affinity ligand surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS) was explored in this work. Multi-protein affinity surfaces were constructed by utilizing antibodies to beta-2-microglobulin, cystatin C, retinol binding protein, transthyretin, serum amyloid P and C-reactive protein. In the initial experiments, all six antibodies were immobilized on a single site (flow cell) on the sensor chip surface, followed by verification of the surface activity via separate injections of purified proteins. After an injection of diluted human plasma aliquot over the antibodies-derivatized surfaces, and subsequent MALDI-TOF MS analysis, signals representing five out of the six targeted proteins were observed in the mass spectra. Further, to avoid the complexity of the spectra, the six proteins were divided into two groups (according to their molecular weight) and immobilized on two separate surfaces on a single sensor chip, followed by an injection of human plasma aliquot. The resulting mass spectra showed signals from all proteins. Also, the convolution resulting from the multiply charged ion species was eliminated. The ability to create such multi-affinity surfaces indicates that smaller-size ligand areas/spots can be employed in the BIA/MS protein interaction screening experiments, and opens up the possibilities for construction of novel multi-arrayed SPR-MS platforms and methods for high-throughput parallel protein interaction investigations.     

Measurement of cortisol,cortisone, prednisolone,dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography–tandem mass spectrometry: Application for plasma,plasma ultrafiltrate,urine and saliva in a routine laboratory

We describe an ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3 min. The limit of quantitation for cortisol and cortisone in plasma was 3.75 nmol/L and linearity extended to 2000 nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6 nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5 nmol/L and for dexamethasone 1 nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC = 0.79 × IA + 31.12 with R² = 0.960 (p < 0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC = 1.06 × HPLC + 9.82, R² = 0.992 (p < 0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.     

Actin protein up-regulated upon infection and development of the filarial parasite, Wuchereria bancrofti (Spirurida: Onchocercidae), in the vector mosquito, Culex quinquefasciatus (Diptera: Culicidae)

Detection and identification of humoral proteins, which are up-regulated in Culex quinquefasciatus upon infection by Wuchereria bancrofti, is important in tracing out the biochemical consequences of the filarial parasite development in the vector mosquito. Analysis of the haemolymph of infected mosquitoes through SDS-PAGE and RP-HPLC showed up-regulation of five proteins of molecular weights 40, 66, 22, 14, and 7-kDa. Among these, only the 40-kDa was unknown and the others were comparable with those already reported as transferrin, attacin, lysozyme, and defensin, respectively. In the present study, the 40-kDa protein up-regulated upon infection was identified as actin through nano-LC-MS/MS analysis. Actin is known to be one of the cytoskeletal proteins up-regulated in the haemolymph, as part of the innate immune system, of Escherichia coli challenged Drosophila melanogaster larvae. For the first time, we have observed an increased level of actin in the haemolymph of W. bancrofti-infected Cx. quinquefasciatus. However, the exact mechanism of actin involvement in the immune system of this mosquito is yet to be studied.     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

Identification of recombinant AtPYL2, an abscisic acid receptor,in E. coli using a substrate-derived bioactive small molecule,a biotin linker with alkyne and amino groups,and a protein cross-linker

Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3′-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).