Automated water analyser computer supported system (AWACSS) Part I: Project objectives, basic technology, immunoassay development, software design and networking

A novel analytical system AWACSS (automated water analyser computer-supported system) based on immunochemical technology has been developed that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration nor pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) (98/83/EC, 1998) and Water Framework Directive WFD (2000/60/EC, 2000), drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The instrument was equipped with remote control and surveillance facilities. The system's software allows for the internet-based networking between the measurement and control stations, global management, trend analysis, and early-warning applications. The experience of water laboratories has been utilised at the design of the instrument's hardware and software in order to make the system rugged and user-friendly. Several market surveys were conducted during the project to assess the applicability of the final system. A web-based AWACSS database was created for automated evaluation and storage of the obtained data in a format compatible with major databases of environmental organic pollutants in Europe. This first part article gives the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part article reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods.     

Hybrid capture II and polymerase chain reaction for identifying HPV infections in samples collected in a new collection medium: a comparison

OBJECTIVE: To compare the performance of human papillomavirus (HPV) DNA detection by polymerase chain reaction (PCR) and Hybrid Capture II (HCII) test (Digene, Gaithersburg, Maryland, U.S.A.) in residual cells left in the collection vials of the DNACitoliq system (Digene Brasil, S?o Paulo, Brazil). STUDY DESIGN: A series of 263 cervical samples collected for liquid-based cytology with the DNACitoliq system was tested for oncogenic HPV types first with HCII and subsequently with PCR. After DNA purification with GFX Genomic Blood DNA Purification Kit (Amersham, Piscataway, New Jersey, U.S.A.), PCR was performed using AmpliTaq Gold DNA polymerase (Applied Biosystems). PGMY09/11 L1 consensus primers and GH20/PCO4 primers for human beta-globin target were coamplified. RESULTS: Altogether, 260 samples were positive for beta-globin, and 3 negative ones were excluded from the analysis. PCR and HCII yielded concordant results in 199 cases (76.5%) (102 positive and 97 negative), with Cohen's kappa of .577 (95% CI .477-.677) and weighted kappa of .733 (95% CI .659-.791). HPV prevalence in different categories of cytologic abnormalities was practically identical with HCII and PCR assays (P=.989). Among the 61 (23.5%) discrepant cases, 28 samples were HCII+/PCR- cases. Of these, 27 of 28 samples showed a low viral load, and 1 had an intermediate viral load. CONCLUSION: The data suggest that residual material from the DNACitoliq system adequately preserves HPV DNA for detection by HCII and PCR, with performance similar to that of specimen transport medium.     

A Trans-acting Factor May Modify Age at Onset in Familial Amyloid Polyneuropathy ATTRV30M in Portugal

Although all familial amyloid polyneuropathy (FAP) ATTRV30M patients carry the same causative mutation, early (<40) and late-onset forms (≥50 years) of FAP may coexist in the same family. However, this variability in age at onset is still unexplained. To identify modifiers closely linked to the TTR locus that may in part be associated with age at onset of FAP ATTRV30M, in particular in a group of very early-onset patients (≤30 years) when compared with late-onset individuals. A clinical genetic study at a referral center comprising a sample of 910 Portuguese individuals includes 589 Val30Met carriers, 102 spouses, and 189 controls from the general population. Haplotype analysis was performed, using eight intragenic single nucleotide polymorphisms (SNPs) at the TTR locus. We compared haplotypes frequency in FAP samples and controls and in parent-offspring pairs using appropriated statistical analysis. Haplotype A was the most common in the general population. Noteworthy, haplotype C was more frequent in early-onset (<40) than in late-onset patients (≥50 years) (p = 0.012). When comparing allelic frequencies of each SNP within haplotype C between “very early” (≤30 years) and late-onset (≥50 years) cases, the A allele of rs72922947 was associated with an earlier onset (p = 0.009); this remained significant after a permutation-based correction. Also, the heterozygous genotype (GA) for this SNP was associated with a decrease in mean age at onset of 8.6 years (p = 0.014). We found a more common haplotype (A) linked to the Val30Met variant and a possible modulatory trans effect on age at onset. These findings may lead to potential therapeutical targets.     

Optimization for peptide sample preparation for urine peptidomics

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.     

Proteome insights into the symbiotic relationship between a captive colony of Nasutitermes corniger and its hindgut microbiome

We analyzed the metaproteome of the bacterial community resident in the hindgut paunch of the wood-feeding ‘higher'' termite (Nasutitermes) and identified 886 proteins, 197 of which have known enzymatic function. Using these enzymes, we reconstructed complete metabolic pathways revealing carbohydrate transport and metabolism, nitrogen fixation and assimilation, energy production, amino-acid synthesis and significant pyruvate ferredoxin/flavodoxin oxidoreductase protein redundancy. Our results suggest that the activity associated with these enzymes may have more of a role in the symbiotic relationship between the hindgut microbial community and its termite host than activities related to cellulose degradation.