Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Effects of a participatory approach,with systematic impact matrix analysis in herd health planning in organic dairy cattle herds

The animal health and welfare status in European organic dairy production does not in all aspects meet the organic principles and consumers’ expectations and needs to be improved. To achieve this, tailored herd health planning, targeted to the specific situation of individual farms could be of use. The aim of this study was to apply herd health planning in a structured participatory approach, with impact matrix analysis, not previously used in this context, in European organic dairy farms and to assess changes in animal health and welfare. Herd health planning farm visits were conducted on 122 organic dairy farms in France, Germany and Sweden. The farmer, the herd veterinarian and/or an advisor took part in the farm discussions. The researcher served as facilitator. Baseline data on the animal health status of the individual farm, collected from national milk recording schemes, were presented as an input for the discussion. Thereafter a systematic impact matrix analysis was performed. This was to capture the complexity of individual farms with the aim to identify the farm-specific factors that could have a strong impact on animal health. The participants (i.e. farmer, veterinarian and advisor) jointly identified areas in need of improvement, taking the health status and the interconnected farm system components into account, and appropriate actions were jointly identified. The researcher took minutes during the discussions, and these were shared with the participants. No intervention was made by the researcher, and further actions were left with the participants. The number of actions per farm ranged from 0 to 22. The change in mortality, metabolic diseases, reproductive performance and udder health was assessed at two time points, and potential determinators of the change were evaluated with linear regression models. A significant association was seen between change in udder health, as measured by the somatic cell count, and country. At the first follow-up, a significant association was also found between change in the proportion of prolonged calving interval and the farmers’ desire to improve reproductive health as well as with an increase in herd size, but this was not seen at the second follow-up. The degree of implementation of the actions was good (median 67%, lower quartile 40%, upper quartile 83%). To conclude, the degree of implementation was quite high, improvement of animal health could not be linked to the herd health planning approach. However, the approach was highly appreciated by the participants and deserves further study.     

Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

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Highlights

• •MS-based clinical assay that accurately determines phospho Rab10 occupancy.
• •Stable isotope labeled phosphopeptide injected as a standard with endogenous tryptic phospho Rab peptide for accurate ratio determination.
• •Determination of pRab levels in neutrophils of Parkinson disease patients.
• •Relevance of pRab levels as marker of PD.

     

Sequence homology and structure predictions of the creatine kinase isoenzymes

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guamidino kinases. A CK framework is defined, consisting of the most conserved sequence blocks, and diagnostic boxes are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

A rapid assay for chloroplast-encoded triazine resistance in higher plants

We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through time and space and can serve as a model system applicable to other gene monitoring needs.     

东亚和东南亚旧石器初期重型工具的类型学：评Movius的分类体系

实践证明,Ｍｏｖｉｕｓ在本世纪４０年代建立的东亚,东南亚旧石器分类体系有严重缺陷。经过厘定,目前在欧洲和非洲通用的吹斫器,手斧,薄刃斧,手镐,球状器和刮削器等分类概念,也适用于东亚,东南亚旧石器初期的重型工具工业。     

Use of comparative reasoning tools for evaluation of the effect of sterilization conditions on fermentations to produce acidic fibroblast growth factor

The effects of various medium sterilization conditions on fermentations of a recombinant, acidic fibroblast growth factor (aFGF) producing Escherichia coli have been studied. Changes in the medium resulting from sterilization were monitored by pH and absorption spectra. This simple experiment provided excellent data for the demonstration of the usefulness of comparative reasoning tools in order to evaluate the effect of sterilization on fermentation performance. The time profiles of the main parameters (e.g., carbon dioxide evolution rate, dissolved oxygen, pH, and aFGF productivity) were simplified into piecewise contiguous linear segments, each of which was sequentially numbered. The length, position, and slope of each tine were then characterized. Application of the comparative reasoning tools confirmed that separate sterilization of the glucose was necessary for the success of the process, despite adding to the cost and complexity. The comparative data analysis also showed that scaleup with longer sterilization holding and cooling times would not be detrimental to aFGF production. (c) 1995 John Wiley & Sons, Inc.     

GENETIC DIVERSITY WITHIN BALTIC SEA POPULATIONS OF NODULARIA (CYANOBACTERIA)1

The determination of the genetic structure of microbial populations has, until recently, required the establishment of many independent clonal cultures for genotypic analysis. In such studies it has been necessary to assume that isolates able to grow in laboratory culture are representative of the full range of diversity within the natural population. In order to test this assumption we used the polymerase chain reaction (PCR) to amplify the intergenic spacer region of the Phycocyanin operon (PC-IGS) from filaments of Nodularia taken both from clonal cultures and from natural populations in the Baltic Sea. Analysis of the nucleotide sequences revealed more variation among 16 cultured isolates than within 23 single filaments sampled from a natural population. As a means of rapidly determining population genetic structure we designed and used mixtures of allele-specific amplification primers in diagnostic PCRs to identify which PC-IGS allele was present in single filaments from natural cyanobacterial assemblages. Using this method, we determined the PC-IGS genotype of 156 filaments from 9 sampling stations throughout the central basin of the Baltic Sea in July 1996. Our results show that two distinct genotypes of Nodularia are present in the population at all stations. Although the two types were present in approximately equal numbers, they were not distributed uniformly.