Evaluation of automated analysis of 15N and total N in plant material and soil

Simultaneous determination of ¹⁵N and total N using an automated nitrogen analyser interfaced to a continuous-flow isotope ratio mass spectrometer (ANA-MS method) was evaluated. The coefficient of variation (CV) of repeated analyses of homogeneous standards and samples at natural abundance was lower than 0.1%. The CV of repeated analyses of ¹⁵N-labelled plant material and soil samples varied between 0.3% and 1.1%. The reproductibility of repeated total N analyses using the automated method was comparable to results obtained with a semi-micro Kjeldahl procedure. However, the automated method gave results which were 3% to 5% higher than those obtained with the Kjeldahl procedure. Since only small samples can be analysed, careful sample homogenization and fine grinding are very important. Evaluation of a diffusion method for preparing nitrate and ammonium in solution for automated ¹⁵N analysis showed that the recovery of inorganic N in the NH₃ trap was lower when the N was diffused from water than from 2 M KCl. The results also indicated that different proportions of the NO₃ ^- and the NH₄ ⁺ in aqueous solution were recovered in the trap after combined diffusion. The method is most suited for diffusing either NO₃ ^- or NH₄ ⁺ alone, but can be used for combined diffusion of the two ions.     

EMAP-Wetlands: A sampling design with global application

The U.S. Environmental Protection Agency (EPA) initiated the Environmental Monitoring and Assessment Program (EMAP) in 1988. The wetland component (EMAP-Wetlands) is designed to provide quantitative assessments of the current status and long-term trends in the ecological condition of wetland resources. EMAP-Wetlands will develop a wetland monitoring network and will identify and evaluate indicators that describe and quantify wetland condition. The EMAP-Wetlands network will represent a probability sample of the total wetland resource. The EMAP sample is based on a triangular grid of approximately 12,600 sample points in the conterminous U.S. The triangular grid adequately samples wetland resources that are common and uniformly distributed in a region, such as the prairie pothole wetlands of the Midwest. However, the design is flexible and allows the base grid density to be increased to adequately sample wetland resources, such as the coastal wetlands of the Gulf of Mexico, which are distributed linearly along the coast. The Gulf sample network required a 49-fold increase in base grid density. EMAP-Wetlands aggregates the 56 U.S. Fish and Wildlife Service's (FWS) National Wetland Inventory (NWI) categories (Cowardin et al. 1979) into 12 functionally similar groups (Leibowitz et al. 1991). Both the EMAP sample design and aggregated wetland classes are suitable for global inventory and assessment of wetlands.The research described in this report has been funded by the U.S. Environmental Protection Agency. This document has been prepared at the EPA Environmental Research Laboratory in Corvallis, OR, through contract No. 68-C8-0006 to Man Tech Environmental Technology, Inc. This paper has been subjected to the Agency's peer and administrative review and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.     

Method for accurate diagnose of lupus erythematosus skin lesions based on microbial rDNA sequencing

This study used microbial rDNA sequencing for accurate detection of systemic lupus erythematosus (SLE) skin lesions. 20 lupus erythematosus dermatology patients and 20 healthy persons were selected as experimental group and control group. Feces and serum of the subjects were sampled in sterile environment. Serum samples were examined for anti dsDNA antibodies. Fecal samples were analyzed by 16 s rDNA high-throughput sequencing, using Illumina MiSeq 2 × 250 sequencing platform. The results suggested that positive rate of anti-dsDNA antibody in serum was significantly higher in the experimental group than the control group. Significant difference of intestinal microbiome was spotted between the two groups in phylum (Firmicutes, Bacteroidetes) and genus level (Lactobacillus, Allobaculum, Lachnospira, Turicibacter, Bifidobacterium). The different intestinal microbiomes existing in healthy people and patients can provide a strong tool for accurate diagnose of lupus erythematosus.     

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

The study was performed to compare real-time PCR after nucleic acid extraction directly from stool samples as well as from samples stored and transported on Whatman papers or flocked swabs at ambient temperature in the tropics. In addition, the possible suitability for a clear determination of likely aetiological relevance of PCR-based pathogen detections based on cycle threshold (Ct) values was assessed. From 632 Tanzanian children <5 years of age with and without gastrointestinal symptoms, 466 samples were subjected to nucleic acid extraction and real-time PCR for gastrointestinal viral, bacterial and protozoan pathogens. Equal or even higher frequencies of pathogen detections from Whatman papers or flocked swabs were achieved compared with nucleic acid extraction directly from stool samples. Comparison of the Ct values showed no significant difference according to the nucleic acid extraction strategy. Also, the Ct values did not allow a decision whether a detected pathogen was associated with gastrointestinal symptoms.     

Design and operation of a completely automated Beckman microsequencer

A unique, efficient, and inexpensive system has been designed and built for the automatic conversion of anilinothiazolinone derivatives extracted from a Beckman spinning-cup sequencer with subsequent on-line high-pressure liquid chromatography separation of the phenylthiohydantoin derivatives. The Auto Converter-Auto Sampler system is controlled by a tape programmer or microprocessor and operates by transfer of the sample from the conversion vial into an HPLC injection loop by nitrogen pressure. Incorporation of a minor programming change on the sequencer allows the introduction of nitrogen vapor into the spinning cup during phenylisothiocyanate coupling. These modifications have resulted in a completely automated subnanomole protein sequencer.     

Human Equilibrative Nucleoside Transporter-3 (hENT3) Spectrum Disorder Mutations Impair Nucleoside Transport,Protein Localization,and Stability

Accumulating evidence reveals that sole mutations in hENT3 cause a spectrum of human genetic disorders. Among these include H syndrome, characterized by scleroderma, hyperpigmentation, hypertrichosis, hepatomegaly, cardiac abnormalities and musculoskeletal deformities, pigmented hypertrichotic dermatosis with insulin-dependent diabetes syndrome, characterized by autoantibody-negative diabetes mellitus and skin deformities, familial Rosai-Dorfman disease, characterized by short stature, familial histiocytosis and sinus histiocytosis with massive lymphadenopathy (SHML), characterized by severe tissue infiltration of immune cells and swollen lymph nodes. hENT3 spectrum disorders share a common mutation and share overlapping clinical manifestations that display many intriguing resemblances to mitochondrial and lysosomal disorders. Although earlier studies identify hENT3 as a mitochondrial and a lysosomal nucleoside transporter, the precise connections between hENT3 and the pathophysiology of these disorders remain unresolved. In this study, we performed functional and biochemical characterization of these mutations in hENT3. We report severe reductions/losses of hENT3 nucleoside transport functions of hENT3 syndrome mutants. In addition to transport alterations, we provide evidence for possible loss of hENT3 functions in all H and pigmented hypertrichotic dermatosis with insulin-dependent diabetes syndromes due to either mistrafficking or altered stability of mutant hENT3 proteins.     

Differences between ranamargarin and other tachykinins in the stimulation of ion transport in frog skin

In frog skin, tachykinins stimulate ion transport by interaction with NK1-like receptors. The structural requirements of the peptide are the presence of the C-terminal sequence Phe-X-Gly-Leu-Met-NH₂ and at least one Pro residue in the N-terminal sequence. In this paper, we demonstrate that the C-terminal amino acid must be amidated but it can be different from Met, and that the sequence cannot be longer or shorter than 11–12 amino acids. Unexpectedly, Ranamargarin (14 amino acids, no Pro residue) increased the short circuit current value by 48 ± 0.3%. On the basis of considerable experimental evidence, we suggest that Ranamargarin interacts with a receptor different from those of other tachykinins.