UK Transfusion Laboratory Collaborative: minimum standards for staff qualifications, training, competency and the use of information technology in hospital transfusion laboratories 2014.

The SHOT Adverse Incident Reporting Scheme has consistently reported an unacceptably high level of errors originating in the laboratory setting. In 2006 an initiative was launched in conjunction with the IBMS, SHOT, RCPath, BBTS, UK NEQAS, the NHSE NBTC and the equivalents in Scotland, Wales and Northern Ireland that led to the formation of the UK TLC. The UK TLC in considering the nature and spread of the errors documented by SHOT concluded that a significant proportion of these errors were most likely to be related to either the use of information technology or staff education, staffing levels, skill mix, training and competency issues. In the absence of any formal guidance on these matters, the UK TLC developed a series of recommendations using the results of two laboratory surveys conducted in 2007 and 2008.

Eribulin mesylate pharmacokinetics in patients with solid tumors receiving repeated oral ketoconazole.

Purpose To study the influence of repeated oral administration of ketoconazole, a potent CYP3A4 inhibitor, on the plasma pharmacokinetics of eribulin mesylate administered by single-dose intravenous infusion. Eribulin mesylate is a non-taxane microtubule dynamics inhibitor that is currently under development in phase I-III trials for the treatment of solid tumors. Experimental design A randomized, open-label, two treatments, two sequences, crossover phase I study was performed in patients with advanced solid tumors. Treatments were given on day 1 and day 15 and consisted of 1.4 mg/m(2) eribulin mesylate alone or 0.7 mg/m(2) eribulin mesylate plus 200 mg ketoconazole on the day of eribulin mesylate administration and the following day. Pharmacokinetic sampling for determination of eribulin plasma concentration was performed up to 144 h following administration of eribulin mesylate. Also safety and anti-tumor activity were determined. Results Pharmacokinetic sampling and analysis was completed in ten patients. Statistical analysis of dose-normalized log-transformed AUC0-∞ and Cmax indicated that single-dose exposure of eribulin was not statistically different when co-administered with ketoconazole (ratio of geometric least square means: 0.95 (90%CI: 0.80-1.12) and 0.97 (90%CI: 0.83-1.12), respectively) in patients with solid tumors. Ketoconazole had no effect on eribulin clearance and elimination half-life. The most frequently reported treatment related adverse events were fatigue and nausea, each reported in 8/12 patients. Seven patients (58.3 %) achieved stable disease as best overall response. Conclusions The results indicate that eribulin mesylate can be safely co-administered with ketoconazole. Drug-drug interactions are not expected with other CYP3A4 inhibitors.

Ribosomal pausing during translation of an RNA pseudoknot.

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal.

The ribosomal frameshift signal in the genomic RNA of the coronavirus IBV is composed of two elements, a heptanucleotide "slippery-sequence" and a downstream RNA pseudoknot. We have investigated the kinds of slippery sequence that can function at the IBV frameshift site by analysing the frameshifting properties of a series of slippery-sequence mutants. We firstly confirmed that the site of frameshifting in IBV was at the heptanucleotide stretch UUUAAAC, and then used our knowledge of the pseudoknot structure and a suitable reporter gene to prepare an expression construct that allowed both the magnitude and direction of ribosomal frameshifting to be determined for candidate slippery sequences. Our results show that in almost all of the sequences tested, frameshifting is strictly into the -1 reading frame. Monotonous runs of nucleotides, however, gave detectable levels of a -2/+1 frameshift product, and U stretches in particular gave significant levels (2% to 21%). Preliminary evidence suggests that the RNA pseudoknot may play a role in influencing frameshift direction. The spectrum of slip-sequences tested in this analysis included all those known or suspected to be utilized in vivo. Our results indicate that triplets of A, C, G and U are functional when decoded in the ribosomal P-site following slippage (XXXYYYN) although C triplets were the least effective. In the A-site (XXYYYYN), triplets of C and G were non-functional. The identity of the nucleotide at position 7 of the slippery sequence (XXXYYYN) was found to be a critical determinant of frameshift efficiency and we show that a hierarchy of frameshifting exists for A-site codons. These observations lead us to suggest that ribosomal frameshifting at a particular site is determined, at least in part, by the strength of the interaction of normal cellular tRNAs with the A-site codon and does not necessarily involve specialized "shifty" tRNAs.

Mutational analysis of the RNA pseudoknot component of a coronavirus ribosomal frameshifting signal.

The genomic RNA of the coronavirus IBV contains an efficient ribosomal frameshift signal at the junction of the overlapping 1a and 1b open reading frames. The signal is comprised of two elements, a heptanucleotide "slip-site" and a downstream tertiary RNA structure in the form of an RNA pseudoknot. We have investigated the structure of the pseudoknot and its contribution to the frameshift process by analysing the frameshifting properties of a series of pseudoknot mutants. Our results show that the pseudoknot structure closely resembles that which can be predicted from current building rules, although base-pair formation at the region where the two pseudoknot stems are thought to stack co-axially is not a pre-requisite for efficient frameshifting. The stems, however, must be in close proximity to generate a functional structure. In general, the removal of a single base-pair contact in either stem is sufficient to reduce or abolish frameshifting. No primary sequence determinants in the stems or loops appear to be involved in the frameshift process; as long as the overall structure is maintained, frameshifting is highly efficient. Thus, small insertions into the pseudoknot loops and a deletion in loop 2 that reduced its length to the predicted functional minimum did not influence frameshifting. However, a large insertion (467 nucleotides) into loop 2 abolished frameshifting. A simple stem-loop structure with a base-paired stem of the same length and nucleotide composition as the stacked stems of the pseudoknot could not functionally replace the pseudoknot, suggesting that some particular conformational feature of the pseudoknot determines its ability to promote frameshifting.

Nitrite-induced deamination and hypochlorite-induced oxidation of DNA in intact human respiratory tract epithelial cells.

No modification of purine or pyrimidine bases was observed when isolated DNA was incubated with 1 mM nitrite at pH 7.4. However, exposure of human bronchial epithelial cells in culture medium at pH 7.4 to nitrite at concentrations of 100 microM or greater led to deamination of purine bases in cellular DNA. Deamination was more extensive in cells exposed to lower extracellular pH values and higher nitrite concentrations. Significant increases in the levels of xanthine and hypoxanthine, putative deamination products of guanine and adenine, respectively, were observed in DNA from nitrite-treated cells but no rise in any base oxidation products such as 8-hydroxyguanine. This pattern of damage suggests that exposure of cells to nitrite (even at pH 7.4) leads to intracellular generation of "reactive nitrogen species" capable of deaminating purines in DNA. In addition, significant DNA strand breakage occurred in nitrite-treated cells. The time course of base damage suggested that the repair of deaminated purine lesions in these cells is slow. By contrast, DNA isolated from cells exposed to hypochlorous acid (HOCl) has significant oxidation of pyrimidine bases and chlorination of cytosine but little oxidation of purines. Exposure of cells to both species (NO(2)(-) plus HOCl) potentiated the oxidative DNA base damage observed but decreased the extent of deamination. We hypothesize that this is due to the formation of nitryl chloride (NO(2)Cl) from reaction of HOCl with *NO(2)(-). The relevance of our observations to events in the stomach and respiratory tract, at sites of inflammation, and in ischemic tissues is discussed.

DNA strand breakage and base modification induced by hydrogen peroxide treatment of human respiratory tract epithelial cells.

Treatment of human respiratory tract epithelial cells with H2O2 led to concentration-dependent DNA strand breakage that was highly-correlated with multiple chemical modifications of all four DNA bases, suggesting that damage is due to hydroxyl radical, OH. However, the major base damage occurred to adenine. Hence, conclusions made about the occurrence and the extent of oxidative DNA damage on the basis only of changes in 8-hydroxyguanine should be approached with caution.

DNA damage in human respiratory tract epithelial cells: damage by gas phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals.

Treatment of human respiratory tract tracheobronchial epithelial cells with gas-phase cigarette smoke led to dose-dependent DNA strand breakage that was highly correlated with multiple chemical modifications of all four DNA bases. The pattern of base damage suggests attack by hydroxyl radicals (OH.). However, by far the most important base damage in quantitative terms was formation of xanthine and hypoxanthine, presumably resulting from deamination of guanine and adenine respectively. Hence, DNA damage by cigarette smoke may involve reactive nitrogen species as well as reactive oxygen species.

Intense oxidative DNA damage promoted by L-dopa and its metabolites. Implications for neurodegenerative disease.

Oxidative DNA damage can cause mutation and cell death. We show that L-DOPA, dopamine and 3-O-methyl-DOPA cause extensive oxidative DNA damage in the presence of H2O2 and traces of copper ions. 8-Hydroxyguanine is the major product. Iron ions were much less effective and manganese ions did not catalyse DNA damage. We propose that copper ion release, in the presence of L-DOPA and its metabolites, may be an important mechanism of neurotoxicity, e.g. in Parkinson's disease and amyotrophic lateral sclerosis.

Demyelination of sciatic nerve in acute experimental allergic neuritis of the rat. A gel electrophoresis investigation.

Acute experimental allergic neuritis (EAN) of Lewis rats was monitored by clinical and electrophysiological tests for 60 days. Sciatic nerve myelin proteins were analyzed by quantitative microgel electrophoresis and electroimmunoblotting at different clinical stages of the disease. The clinical severity of EAN and the demyelination as measured by electrophysiological tests and myelin protein concentrations in sciatic nerve did not correspond during the course of the disease. Demyelination reached its peak after partial clinical recovery and was still present on day 60. The major peripheral nervous system (PNS) myelin proteins P0, P1 and P2 decreased at different rates during the course of the disease, indicating possible differences in their proteolytic degradation. Treatment with Freund's complete adjuvant (CFA) alone resulted in alterations of membrane and myelin protein patterns challenging the widely held belief that pure CFA is inert with regard to demyelination.

6-hydroxydopamine increases hydroxyl free radical production and DNA damage in rat striatum.

Oxidative damage is considered to be an important factor of 6-hydroxydopamine (6-OHDA) toxicity. To address this issue, microdialysis probes were implanted into the striatum of Wistar rats and perfused with 6-OHDA. Salicylate was included in the perfusion fluid to measure 2,3-dihydroxybenzoic acid (2,3-DHBA) as a marker of hydroxyl radical formation using HPLC with electrochemical detection. Additionally, striatal tissue was analysed for DNA base alterations using gas chromatography-mass spectrometry. 6-OHDA administration resulted in a rapid and substantial 6.6-fold increase in 2,3-DHBA formation and also increased levels of the modified DNA bases 5-hydroxycytosine, hypoxanthine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. Hydroxyl radical formation and DNA base alterations are early phenomena of 6-OHDA toxicity and provide clues to the processes that may be involved in the initiation of cell death in Parkinson's disease.

Determination of oxidative DNA base damage by gas chromatography-mass spectrometry. Effect of derivatization conditions on artifactual formation of certain base oxidation products.

GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140 degrees C were raised 8-fold compared to derivatization at 23 degrees C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140 degrees C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured. This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23 degrees C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O2 during the derivatization process.

Evaluation of the pro-oxidant and antioxidant actions of L-DOPA and dopamine in vitro: implications for Parkinson's disease.

The antioxidant and pro-oxidant properties of L-DOPA and dopamine were investigated in vitro. Both compounds inhibited the peroxidation of ox-brain phospholipids, with IC50 values of 8.5 microM for dopamine and 450 microM for L-DOPA. Dopamine and L-DOPA reacted with trichloromethyl peroxyl radicals (CCl3O2.) with rate constants of 2.1 x 10(7)M-1s-1 and 1.3 x 10(7)M-1s-1 respectively. The effects of dopamine and L-DOPA on iron ion-dependent hydroxyl radical generation from H2O2 were complex. In general, low concentrations stimulated OH. formation in the presence of ferric-EDTA and, in the case of L-DOPA, ferric-ADP and ferric citrate chelates. Both compounds also reacted with superoxide radical and hypochlorous acid. The products of the reaction with HOCl could still inhibit alpha 1-antiproteinase and appear to be 'long lived' chloramine-type oxidizing species. Our results suggest that L-DOPA and dopamine might have a complex mixture of pro- and anti- oxidant effects, which could contribute to tissue damage due to oxidative stress in Parkinson's disease and other neurological disorders.

The cerebellum's role in reading: a functional MR imaging study.

BACKGROUND AND PURPOSE: Long considered to have a role limited largely to motor-related functions, the cerebellum has recently been implicated as being involved in both perceptual and cognitive processes. Our purpose was to determine whether cerebellar activation occurs during cognitive tasks that differentially engage the component processes of word identification in reading. METHODS: Forty-two neurologically normal adults underwent functional MR imaging of the cerebellum with a gradient-echo echo-planar technique while performing tasks designed to study the cognitive processing used in reading. A standard levels-of-processing paradigm was used. Participants were asked to determine whether pairs of words were written in the same case (orthographic processing), whether pairs of words and non-words rhymed with each other, respectively (phonologic assembly), and whether pairs of words belonged to the same category (semantic processing). Composite maps were generated from a general linear model based on a randomization of statistical parametric maps. RESULTS: During phonologic assembly, cerebellar activation was observed in the middle and posterior aspects of the posterior superior fissure and adjacent simple lobule and semilunar lobule bilaterally and in posterior aspects of the simple lobule, superior semilunar lobule, and inferior semilunar lobule bilaterally. Semantic processing, however, resulted in activation in the deep nuclear region on the right and in the inferior vermis, in addition to posterior areas active in phonologic assembly, including the simple, superior semilunar, and inferior semilunar lobules. CONCLUSION: The cerebellum is engaged during reading and differentially activates in response to phonologic and semantic tasks. These results indicate that the cerebellum contributes to the cognitive processes integral to reading.

Effect of inhibin-like activity on LH-RH-stimulated release of FSH by pituitary glands from female rats in vitro.

During long-term incubation of pituitary glands from intact female rats in the presence of inhibin-like activity, LH-RH-stimulated release of FSH becomes inhibited after 4 h of incubation. However, at the same time inhibition of basal FSH release is included. Therefore, glands were at first incubated for 4 h in the presence of inhibin-like activity to block basal release completely and thereafter LH-RH was added to the medium. It was found that LH-RH still could stimulate FSH release, despite the continuous presence of inhibin-like activity. This means that LH-RH-stimulated release of FSH could be investigated separately from basal release. Using this way of incubation, it was found that part of the action of LH-RH on FSH release was independent of protein synthesis. Also part of LH-RH-stimulated FSH release was independent of the presence of extracellular Ca2+. Furthermore it was found that LH-RH, when added after 4 h of incubation did stimulate FSH synthesis, in the presence as well as absence of inhibin-like activity. The present results indicate that LH-RH-stimulated release of FSH is not affected by inhibin-like activity. Complete inhibition of basal release and synthesis of FSH does not prevent LH-RH from stimulating FSH release and synthesis. It is suggested that two separate releasing mechanisms for FSH could exist in the pituitary gland.

Results of the screening program for congenital hypothyroidism in Berlin (1978-1995).

Screening for congenital hypothyroidism for all newborns of the former western parts of the city of Berlin was instituted in 1978 by determination of the TSH levels in dried filter paper blood spots of the 3rd to 5th day of life. Since 1991 the newborns of the former eastern parts have been also included in the same screening program. From 1978 to 1995 a total number of 104 newborns with permanent congenital hypothyroidism have been detected resulting in a prevalence of congenital hypothyroidism of 1:3800. The etiological diagnosis of hypothyroidism was made by imaging studies (ultrasonography or 99mTc scintigraphy) and assessment of serum thyroglobulin and thyroid hormone levels. Using this approach in 37 children (30 female, 7 male) the diagnosis of athyrosis, in 20 children (15 female, 5 male) the diagnosis of ectopy and in 21 children (18 female, 3 male) the diagnosis of thyroid hypoplasia was made, 16 children (8 female, 8 male) had a normally sized gland and 4 (1 female, 3 male) had congenital goiter. In 86% of all patients the age at onset of thyroxine (L-T4) replacement therapy was 8 or 9 days of life (3-42 days) and the median initial L-T4 dose was 14 micrograms/kg/day (10-16 micrograms/kg/day). The intellectual outcome of 77 children (2-16 years) was studied and normal scores for the intelligence (IQ) and developmental (DQ) quotients were found in 71 (92%). Outcome was not correlated to the age at onset of treatment, the initial dose and the severity of hypothyroidism, but there was a positive correlation of the socioeconomic status of the family and the IQ of the patients. The results of the screening program in Berlin document that an early and efficient thyroxine replacement can normalize the intellectual outcome of patients with congenital hypothyroidism independent of the severity of the disease as assessed by the residual thyroid function detectable at diagnosis.

The possibility of school milk as a vehicle for fluoride: epidemiological, organisational and legal considerations.

The aim of this study was to investigate the epidemiological, organisational and legal considerations of using school milk as a vehicle for fluoride. The background to the work was the need to develop alternative methods of fluoride supplementation for high risk groups living in areas where water fluoridation is unlikely. St Helens in Merseyside was chosen as the study area, where 23 per cent of 4-year-old children were identified as having a high caries experience (dmft > or = 4). It was found that 65 per cent of 3 and 4-year-old children attend local authority educational facilities, where milk is provided on a daily basis for children aged 2 to 7 years. This group included 62 per cent of the 4-year-old group with high caries experience. The legal aspects of adding fluoride to milk were found to be complex but it was concluded that there may be considerable potential for using school milk as a vehicle for fluoride, and a large-scale study is now planned.

Referral of child patients from the Community Dental Service to the General Dental Service: one year's experience.

The Community Dental Service has been encouraged to refer regular attending patients to General Dental Practice whilst maintaining a monitoring system to ensure that referred patients do not 'fall through the net'. The referral and monitoring system run by Halton Health Authority for one year is described. During this period 112 motivated patients were identified as being suitable for referral to general practice. Of these, 85 agreed to referral and 27 refused. Of those referred, 69 subsequently attended but 16 failed to attend and 14 of these did not respond to a further recall by the CDS. Far from acting as a safety net service the CDS may, in pursuing this referral policy, be actually increasing the number of irregular attenders in some localities and for this reason Halton is reviewing the speed with which the referral policy is being implemented.

The identification of agreed criteria for referral following the dental inspection of children in the school setting.

AIM: To clarify the function of the school based dental inspection. OBJECTIVE: For representatives of the Community Dental Service, General Dental Service and Hospital Dental Service to identify an agreed set of criteria for the referral of children following school dental inspection. DESIGN: Qualitative research methodology used to establish a consensus for the inclusion of referral criteria following dental screening. SETTING: Ellesmere Port, Cheshire, England. MATERIALS: A Delphi technique was used to establish a consensus amongst the study participants on the inclusion of nine possible criteria for referral following dental screening. All participants scored each criterion in the range 1-9, with a score of 1 indicating that referral of individuals with the condition should definitely not take place, and a score of 9 indicating referral should definitely take place. Referral criteria were accepted only if they achieved a group median score of 7 or more, with an interquartile range of three scale points, with the lower value being no less than 7. RESULTS: Four of the nine possible criteria met the agreed group standard for inclusion: 'Sepsis', 'Caries in the secondary dentition', 'Overjet > 10 mm', and 'Registered & caries in the permanent dentition'. CONCLUSION: It is possible to agree clear criteria for the referral of children following the school dental inspection.

A clinical minimum data set for primary dental care.

OBJECTIVE: To achieve consensus within primary dental care on the contents of a clinical minimum data set to measure oral health status. DESIGN: Using the Delphi process a simple random sample of 30 LDCs and 10 CDS services in England were asked to rank a list of existing clinical indicators in order of their perceived importance as a means of measuring oral health. A nominated panel representing the stakeholder organisations of primary dental care reviewed this ranking and identified a core group of clinical indicators to be included in a clinical minimum data set. RESULTS: An 80 percent response rate to the Delphi process was achieved. Consensus was reached on a core group of 10 indicators, which can provide information on patient's perceptions of pain, function and appearance, and professional measurements of caries, teeth present, periodontal disease, oral sepsis, presence of mucosal pathology and tooth wear. CONCLUSIONS: A representative sample of primary care dentists in England and the key representative organisations of primary dental care achieved consensus on the contents of a clinical minimum data set to record oral health status in primary dental care. This is a first step in standardising the measurement of oral health status across primary care.