Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice.

Mode of action hypothesis testing in chemical safety assessments using metabolomics as supporting evidence: Phenobarbital and cyclobutrifluram metabolomics profile comparison.

In long term rodent studies administering Cyclobutrifluram (TYMIRIUM® Technology), a new agrochemical, there was a slight elevation of incidence of hepatocellular carcinomas in male CD-1 mice that was within the historical control range but appeared to be dose responsive. Cyclobutrifluram's ability to activate mouse constitutive androstane receptor (CAR) mediated gene transcription was confirmed in vitro, therefore a 28-day dietary toxicity study was conducted in vivo in male CD-1 mice to assess the CAR activation mode of action hypothesis of Cyclobutrifluram along with phenobarbital, a known CAR activator. In addition to other end points comprehensive (polar and lipidomic) hybrid metabolomics analyses were performed on terminal plasma and liver samples following 2-, 7- and 28-days dietary exposure to cyclobutrifluram and phenobarbital. The data generation and quality assessments were performed in line with the principles of the MEtabolomics standaRds Initiative in Toxicology (MERIT).First the full annotated feature set was used to compare the metabolomic changes induced by the administration of the two test substances using Shared and Unique Structures plots. This gave a comprehensive overview of the similarity of the two effect profiles showing good correlation and demonstrated that no other, alternative effect signatures were detected. Then the phenobarbital induced differentially abundant metabolites were selected, compared to the literature and their direction of change was assessed in cyclobutrifluram profiles, finding good agreement. Both approaches concluded that the metabolomics data supports the CAR activation hypothesis. Comparison of the metabolomic effect profiles can be a line of evidence in mode of action hypothesis testing in the chemical risk assessment process.

Validation and comparison of three brief depression scales in an elderly Chinese population.

The study aimed to establish the diagnostic accuracy of the Geriatric Depression Scale (GDS), the Even Briefer Assessment Scale for Depression (EBAS DEP), and the single question test for depression in our elderly Chinese population, and to determine if any one instrument was to be preferred. Ninety-eight community-living, socially active and non-depressed elderly and 75 patients diagnosed with depression were administered the three depression scales. Receiver operating characteristics (ROC) were employed to determine the optimal cut-off scores for the GDS and EBAS DEP, and the diagnostic performance of all three instruments were then compared. ROC analysis indicated an optimal cut-off score of 4 and above for the 15-item GDS, with a sensitivity of 84.0% and a specificity of 85.7%, while the EBAS DEP had 77.3% sensitivity and 89.8% specificity at the optimal cut-off score of 3 and above. The sensitivity and specificity of the single question were 64.0% and 94.9%, respectively. The non-parametric test of the areas-under-the-curve showed no significant difference between the diagnostic performances of the GDS and the EBAS DEP; visually, however, the ROC plot of the GDS was superior. The GDS, the EBAS DEP, and the single question were all valid screening tools for depression in the elderly Chinese population. For busy physicians, there is rationale to first use the single-question test, supplemented where necessary with either the GDS or the EBAS DEP, as an efficient diagnostic strategy for identifying depression amongst older Chinese patients.

Diagnostic performance of two mental status tests in the older chinese: influence of education and age on cut-off values.

AIMS: To (1) establish the clinical usefulness of the 10-item Abbreviated Mental Test (AMT) and the 18-item Chinese Mini-Mental Status Examination (CMMSE) for detecting cognitive impairment associated with dementia in the elderly Chinese; (2) determine how the tests' optimal cut-off scores varied with the patients' educational level and age; and (3) evaluate which was the more accurate test. METHODS: 151 cognitively-healthy, community dwelling elderly Chinese subjects and 95 elderly Chinese outpatients with dementia were administered the AMT and CMMSE. Receiver-Operating Characteristic (ROC) analysis was used to determine the tests' optimal cut-off scores for each of the education-by-age subgroups and their areas-under-the-curve were compared non-parametrically to evaluate which test was more accurate. RESULTS: Both the AMT and CMMSE could identify cognitive impairment accurately, but higher cut-off values were necessary for the younger and more educated cohort, while lower values were adequate for the older and less educated subgroup. The AMT appeared to reach a ceiling effect in the more educated categories. The diagnostic accuracies of the two instruments were statistically equivalent; there was a trend, however, for the CMMSE to be performing better in the more educated subgroups. CONCLUSIONS: To maximise the diagnostic efficiency of these two clinically useful mental status tests, it is important to adjust their cut-off scores for the patients' education and age. Though no clear superiority of either instrument was established in this study, we recommend the AMT for patients with 0-6 years of education, whereas for those with greater levels of literacy, we think it better to administer the CMMSE.

Dementia in the hospitalized elderly--a study of 100 consecutive cases in Singapore.

AIM: This survey aims to study the medical, functional and social aspects of hospitalized elderly patients with dementia. METHODS: The case records of 100 patients with dementia admitted consecutively to our department were systematically reviewed to extract the patients' demographic data, presenting features, underlying cause(s) of dementia, functional disabilities and carers' difficulties. RESULTS: Eighty per cent of the patients were 75 years of age and above and 44% were males. In this cohort 55% had vascular dementia, with Alzheimer's disease being the next most common (40%). The overwhelming majority had marked cognitive impairment as assessed by the Abbreviated Mental Test and worsening degrees of dementia were significantly associated with increasing disabilities in both mobility and activities of daily living (ADL). Fifty-seven families reported coping difficulties and this group had significant correlations with the respective patients' bladder and bowel incontinence as well as worsening states of ambulatory and ADL function. CONCLUSION: Patients with dementia present with complex problems and management requires attention not only to patients' medical problems and rehabilitation strategies but also to education, emotional support and help for their frequently stressed out carers.

Burden of caregiving in mild to moderate dementia: an Asian experience.

This survey sought to determine (a) the prevalence of carer stress in patients with mild to moderate dementia, (b) whether caregiver burden was already associated with plans to institutionalize patients, and (c) which patient-related and caregiver-related variables best predicted caregiver burden. The principal caregivers of 93 Asian patients with mild to moderate dementia attending an outpatient cognitive assessment clinic were interviewed via a structured questionnaire that focused upon (a) patient-related variables such as their behavioral and functional abnormalities; and (b) caregiver-related variables such as whether they were having problems looking after the patients, the duration of their caregiving, their associated feelings of anger and/or depression, and their financial status as well as intentions to institutionalize patients. Forty-nine percent of caregivers reported problems in looking after the patients, and their perception of difficulties was significantly associated with institutionalization plans for the patients. Logistic regression analysis using a forward variable selection procedure showed two of the patients' behavioral abnormalities (repetition, agitation) and one of their functional impairments (urinary incontinence) as well as the carers' depressed feelings to be predictive of the carers' problematic status, explaining 40% of the variance. It is important that even in the early stages of dementia, the medical assessment also evaluate behavioral, functional, and social dimensions of the illness, so that appropriate interventions can be implemented to reduce caregiving burden and delay institutionalization.

Physical mapping of a collection of Mael-generating amber mutations in the beta gene of Escherichia coli RNA polymerase and the functional effect of internal deletions constructed through their manipulation.

We report the fine mapping of 55 of our 95 amber mutations in the beta gene of Escherichia coli RNA polymerase by virtue of the unique MaeI restriction sites created by this subset of nonsense mutations (i.e. CTAG, where the amber codon is underlined). [The full data are reported in Supplementary Publication SUP 50181 (12 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8-10.] The CTAG mutations, which have been positioned to within approx. 9-60 bp, are distributed almost along the entire length of the rpoB gene, the one exception being the interval 400-499. The lack of amber fragments for mutations within the 5' approx. 265 codons suggests lability of the extreme N-terminal region; further potential destabilizing 'signals' may be present in the non-conserved 'spacer' regions. The locations of four of the eleven rpoB amber mutations that are strongly polar on expression of the downstream rpoC gene have been determined through a combination of MaeI mapping, PCR amplification and DNA sequencing. Surprisingly, one such mutant carries two tandem CTAG sites but is viable with three of the nonsense suppressors tested. These polar amber sites define three different amino acids (Gln-31, Gln-83 and Trp-183) that fall within three sequence-conservation blocks in the N-terminal region. Six of the MaeI/Am (where MaeI/Am is an amber mutation generating a MaeI restriction site) rpoB alleles (Gln-83, Gln-276, Gln-327, Gln-618, Gln-649 and Trp-183) have been used to generate small in-frame deletions (31-100 codons) within conserved and non-conserved regions of the beta gene, and the properties of these deletion variants were assessed in vivo. The smallest deletion reported in this study removes 31 amino acids from the middle of a region common to the eubacterial/chloroplast subgroup of beta homologues, and our results strongly suggest beta(deltaQ618-Q649) is assembled into a holoenzyme form capable of transcriptional initiation in vivo.

In vivo cloning of a carboxy-terminal rpoB allele which confers altered transcriptional properties.

A 3'-terminal mutation of the gene encoding the beta subunit of Escherichia coli RNA polymerase was isolated using an in vivo polA(Ts) technique. Cloning of the allele was monitored by virtue of the fact that the deletion delta(rpoB)1570-1 resulted in an altered-size restriction fragment. DNA sequencing confirmed the predicted nature and location of the mutation: delta(rpoB)1570-1 involved an in-frame deletion of 186 bp (62 codons) encoding amino acid residues 967-1028. The phenotype conferred by delta(rpoB)1570-1 is discussed with respect to conserved domains within the beta polypeptide.

'Stop-codon-specific' restriction endonucleases: their use in mapping and gene manipulation.

Certain restriction endonucleases recognise target sequences that contain the stop triplet TAG and are commonly either 4 or 6 bp in length. Interestingly, these restriction targets do not occur at the frequency expected on the basis of base composition and size. For example, the tetranucleotide MaeI recognition sequence (CTAG) occurs considerably less commonly (5-8-fold) in the genome of Escherichia coli (and many other eubacteria) than expected from mononucleotide frequencies. This surprising rarity is particularly evident in protein-encoding genes and is largely dictated by codon usage. Thus, amber (TAG) nonsense mutations frequently give rise to novel MaeI (CTAG) sites which are unique within a translated region. Such amber/MaeI sites, whether arising spontaneously or created in vitro by site-directed mutagenesis, act as a useful physical marker for the presence of the nonsense mutation and are a convenient startpoint for a range of diverse procedures. These features provide a useful supplement to protein engineering methods which use nonsense suppression to mediate amino acid replacements.

Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase.

We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions. The 1342-amino acid-long beta subunit is alternatively cleaved at Arg903 or Lys909. The cleavage occurs adjacent to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria. In E. coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function. Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis. The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue 1000), which can be used for anchoring the catalytically active enzyme on a solid support. We also report the identification of a secondary trypsin cleavage at Arg81 of the beta' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts.