Developing behavioural indicators for intellectual functioning and adaptive behaviour for ICD-11 disorders of intellectual development.

BACKGROUND: We present the work conducted to arrive at deriving behavioural indicators that could be used to guide clinical judgement in determining the presence and severity of deficits in intellectual functioning and adaptive behaviour for the purpose of making a diagnosis of disorders of intellectual development. METHODS: An interdisciplinary expert panel provided guidance in developing behavioural indicators for intellectual functioning. A national dataset of adaptive behaviour on a sample of individuals with a diagnosis of intellectual disability was used to develop the behavioural indicators for the adaptive behaviour. The adaptive behaviour data were analysed using a cluster analysis procedure to define the different severity groupings by chronological age groups. RESULTS: We present a series of tables containing behavioural indicators across the lifespan for intellectual functioning and adaptive behaviour, including conceptual, social and practical skills. These tables of behavioural indicators have been proposed for use in the clinical version of the 11th revision of the International Classification of Diseases and Related Health Problems (ICD-11) to be published by the World Health Organization. CONCLUSIONS: The proposed behavioural indicators for disorders of ID described in the present article and to be included in the ICD-11 Clinical Descriptions and Diagnostic Guidelines are put forth to assist professionals in making an informed clinical decision regarding an individual's level of intellectual functioning and adaptive behaviour for the purpose of making a determination about the presence and severity of disorders of ID.

Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c.

BACKGROUND: B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer. METHODS: MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3'-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients). RESULTS: We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3'-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts. CONCLUSIONS: We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.

Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection.

A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.

Polymorphism in the pertussis toxin promoter region affecting the DNA-based diagnosis of Bordetella infection.

The pertussis toxin (PT) promoter region is a frequently used target for DNA-based diagnosis of pertussis and parapertussis infections. The reported polymorphism in this region has also allowed discrimination of species in mixtures with several Bordetella species by their specific PCR amplicon restriction patterns. In the present study, we investigated the degree of polymorphism in order to confirm the reliability of the assay. Five different sequence types of the amplified 239- or 249-bp region were found among the 33 Bordetella pertussis, B. parapertussis, and B. bronchiseptica American Type Culture Collection reference strains and patient isolates analyzed. According to the sequences that were obtained and according to the PT promoter sequences already available in the databases, restriction enzyme analysis with TaqI, BglI, and HaeII, which gave four different patterns, can be performed to reliably identify B. pertussis, B. parapertussis, and B. bronchiseptica.

Aging in Pinus sylvestris L. seeds: changes in viability and lipids.

Aging of Scots pine seeds (Pinus sylvestris L.) leads to changes in seed quality, such as loss of germinability, delayed growth and abnormality in developing seedlings. The knowledge of biochemical changes responsible for these aging processes is plentiful in some seeds, which are of world-wide interest, but for pine seeds these studies are rare. The aim of the present study, was to analyse pine seeds of varying ages in order to identify biochemical changes occurring in aged pine seeds, and to see if a correlation existed between these results and traditionally used seed-quality parameters, such as germinability and electrolyte leakage.

Analyses of secondary structures in DNA by pyrosequencing.

A common problem in conventional DNA sequencing is the occurrence of DNA sequence compressions during gel electrophoresis, leading to misreading of the sequence. These compressions are usually due to secondary structures in the DNA fragment. In this study, we present a non-gel-based DNA sequencing technique that facilitates analysis of such DNA regions. A part of the polymorphic pertussis toxin promoter region in five different Bordetella species was successfully resolved by the new technique. The obtained sequence data revealed four related palindromic sequences. The ability of different DNA polymerases to read through such secondary structures is also described.

Use of multiple competitors for quantification of human immunodeficiency virus type 1 RNA in plasma.

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.

Quantification of Bordetella pertussis in clinical samples by colorimetric detection of competitive PCR products.

Quantification of microorganisms is an important part of the normal diagnostic work of a clinical microbiology laboratory. Traditionally the diagnosis of pertussis is subject to a yes or no approach with no quantitative dimension. This can, however, be of interest as a factor when judging the risk of a patient spreading the bacterium and as a research tool. The aim of the present study was to develop a PCR-based quantitative assay for Bordetella pertussis DNA in clinical nasopharyngeal aspirates by combining a quantitative PCR with a colorimetric detection principle, DIANA (detection of immobilised amplified nucleic acid). A competitor to the PCR target sequence in IS-481, containing a lac-operator, was constructed and calibrated, and a test protocol prepared. A total of 46 clinical nasopharyngeal aspirates, previously diagnosed using a standard nested PCR assay and quantified by culture, were analysed by the quantitative PCR. The method showed acceptable precision and accuracy considering that it estimates the total number of bacterial genomes while culture detects viable bacteria. Recognised advantages were the simple colorimetric detection, the inborn indication of a working PCR assay, and the possibility of obtaining results even when partial inhibition of the PCR assay was seen. In addition, the quantitative PCR result can be obtained within one day compared to 3-10 days for culture. The present results and the qualities of the quantitative PCR suggest that this assay will be a useful complement in routine diagnostics and in research.

Parental presence during induction of anesthesia. A randomized controlled trial.

BACKGROUND: To determine whether parental presence during induction of anesthesia is an effective preoperative behavioral intervention, a randomized controlled trial with children undergoing outpatient surgery was conducted. METHODS: Eighty-four children were randomly assigned to a parent-present or parent-absent group. Using multiple behavioral and physiologic measures of anxiety, the effect of the intervention on the children and their parents was assessed. Predictors for the response to the intervention were examined using multivariate linear regression analysis. RESULTS: When the intervention group (parent-present) was compared to the control group (parent-absent), overall there were no significant differences in any of the behavioral or physiologic measures of anxiety tested during induction of anesthesia. Using the child's serum cortisol concentration as the outcome, parental presence, the child's age and baseline temperament, and trait anxiety of the parent, were identified as predictors of the child's anxiety during induction. Analysis of variance demonstrated that three groups showed diminished cortisol concentrations with parental presence: children older than 4 yr (P = 0.001), children whose parent had a low trait anxiety (P = 0.02), and children who had a low baseline level of activity as assessed by temperament (P = 0.05). CONCLUSIONS: Children who were older than 4 yr or those with a parent with a low trait anxiety or who had a low baseline level of activity/temperament benefited from parental presence during induction.

Biological monitoring of hexachloroethane.

A small group (n = 12) of military white smoke munition workers provided blood plasma during a production break (S I) and after five weeks' production (S II) of a hexachloroethane (HCE)/titanium dioxide formula. Plasma was also obtained from a sex and age matched control group (n = 12) and a group (n = 13) of previously HCE-exposed workers, respectively. HCE in plasma (P-HCE) was determined with gas chromatography and electron capture detection. No HCE was found in the plasma samples from the two control groups. In the HCE exposed group the mean (+/- SD) P-HCE level increased almost two orders of magnitude from S I (0.08 +/- 0.14 microgram/l) to S II (7.30 +/- 6.04 micrograms/l) despite efforts to minimize the internal dose. Biological monitoring of HCE could be useful in occupational hygiene.

World Health Organization international intercalibration study on dioxins and furans in human milk and blood.

Under the sponsorship of the World Health Organization (WHO), an interlaboratory calibration on the analysis of PCDD/PCDFs in human milk and blood was carried out which included 19 laboratories from 14 countries. The study design involved the analysis of three samples of each matrix in triplicate. Selected samples were spiked with native standards of certain 2,3,7,8-substituted congeners at concentrations known only to WHO staff. The study design resulted in approximately 4000 individual pieces of PCDD/PCDF data generated by a variety of analytical methods, at various concentrations, and by laboratories of widely different experience. This was, by considerable margin, the largest study which allowed for the direct comparison of laboratory and method performance. The results of statistical analysis of this data base addresses the effect on data quality of clean up methods, instrumental methods, analyte concentration, laboratory QA programs, and laboratory experience. The study has shown that the laboratory is the single most important determinant of data precision and accuracy. The method of analyte enrichment (sample clean up), analyte measurement [gas chromatography/mass spectrometry (GC/MS) protocol], and analyte concentration have weaker correlations with data quality.

Dioxins and dibenzofurans in blood and adipose tissue of Agent Orange-exposed Vietnam veterans and matched controls.

Vietnam veterans who were heavily exposed to Agent Orange exceeded matched control subjects in both blood and adipose tissue levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but not in the levels of the 12 other 2,3,7,8-substituted dioxins and dibenzofurans that were detected. Since only TCDD among these compounds was present in Agent Orange but all are present in the population of the industrialized world, it is likely that the elevated TCDD levels arose from wartime exposure. The high correlation (r = +.89) of blood with adipose tissue level suggests that there may be a mobile equilibrium between them and that blood measurement could replace adipose tissue measurement of TCDD levels, making the collection of human data less invasive.

Assessment of human exposure to polychlorinated dibenzofurans and dioxins.

Fires, explosions and other accidents in polychlorinated biphenyl (PCB)-filled equipment can result in possible exposure of firemen, cleaning personnel and regular workers. Inhalation, dermal exposure and ingestion are the possible routes of exposure. An indirect assessment of the exposure can be made by analyses of wipes, air and water samples and clothes. A direct assessment of exposure can be made by analyses of blood samples, adipose and other tissue samples, feces and bile.

Polychlorinated biphenyls, dibenzofurans and quaterphenyls in toxic rice-bran oil and in the blood and tissues of patients with PCB poisoning (Yu-Cheng) in Taiwan.

A mass outbreak of poisoning occurred in central Taiwan in 1979 due to the ingestion of rice-bran oil contaminated with polychlorinated biphenyls (PCBs), dibenzofurans (PCDFs) and quaterphenyls (PCQs). The incident was called PCB poisoning or Yu-Cheng in Taiwan. The major PCB and PCDF congeners in the toxic oil and in the blood and tissues of the poisoned patients were characterized by gas chromatography and gas chromatography-mass spectrometry using highly efficient glass capillary columns. The levels of toxic agents in the rice oil samples collected from the factory and school cafeterias and the families of the poisoned patients are in the range of 53 to 99 ppm, 0.18 to 0.40 ppm and 25 to 53 ppm for PCBs, PCDFs, and PCQs, respectively. The blood samples of 165 patients collected 9 to 18 months after the onset of poisoning contained 10 to 720 ppb of PCBs, with a mean value of 38 ppb. The blood samples of 10 patients collected 9 to 27 months after poisoning contained 0.02 to 0.20 ppb of PCDFs. Comparative rates of elimination of some PCB congeners from the blood of patients were studied. Various tissues from a patient who died 2 years after poisoning were analyzed for PCBs, PCDFs and PCQs. The intestinal fat contains the highest level of PCBs, while the liver contains the highest concentration of PCDFs. The PCB congeners retained in the tissues either do not have adjacent unsubstituted carbon atoms or have a pair at ortho-meta positions of the biphenyl ring. The major PCDF congeners retained in the tissues were 1,2,3,4,7,8-hexachloro-DF, 2,3,4,7,8-pentachloro-DF and 1,2,4,7,8-pentachloro-DF. The former two congeners, especially 2,3,4,7,8-pentachloro-DF, are very toxic PCDFs; they may play important roles in the etiology of Yu-Cheng.