Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation.

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced microRNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these pro-differentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.

Serial murder by healthcare professionals.

The prosecution of Charles Cullen, a nurse who killed at least 40 patients over a 16-year period, highlights the need to better understand the phenomenon of serial murder by healthcare professionals. The authors conducted a LexisNexis search which yielded 90 criminal prosecutions of healthcare providers that met inclusion criteria for serial murder of patients. In addition we reviewed epidemiologic studies, toxicology evidence, and court transcripts, to provide data on healthcare professionals who have been prosecuted between 1970 and 2006. Fifty-four of the 90 have been convicted; 45 for serial murder, four for attempted murder, and five pled guilty to lesser charges. Twenty-four more have been indicted and are either awaiting trial or the outcome has not been published. The other 12 prosecutions had a variety of legal outcomes. Injection was the main method used by healthcare killers followed by suffocation, poisoning, and tampering with equipment. Prosecutions were reported from 20 countries with 40% taking place in the United States. Nursing personnel comprised 86% of the healthcare providers prosecuted; physicians 12%, and 2% were allied health professionals. The number of patient deaths that resulted in a murder conviction is 317 and the number of suspicious patient deaths attributed to the 54 convicted caregivers is 2113. These numbers are disturbing and demand that systemic changes in tracking adverse patient incidents associated with presence of a specific healthcare provider be implemented. Hiring practices must shift away from preventing wrongful discharge or denial of employment lawsuits to protecting patients from employees who kill.

Serial murder by healthcare professionals.

The prosecution of Charles Cullen, a nurse who killed at least 40 patients over a 16-year period, highlights the need to better understand the phenomenon of serial murder by healthcare professionals. The authors conducted a LexisNexis search which yielded 90 criminal prosecutions of healthcare providers that met inclusion criteria for serial murder of patients. In addition we reviewed epidemiologic studies, toxicology evidence, and court transcripts, to provide data on healthcare professionals who have been prosecuted between 1970 and 2006. Fifty-four of the 90 have been convicted; 45 for serial murder, four for attempted murder, and five pled guilty to lesser charges. Twenty-four more have been indicted and are either awaiting trial or the outcome has not been published. The other 12 prosecutions had a variety of legal outcomes. Injection was the main method used by healthcare killers followed by suffocation, poisoning, and tampering with equipment. Prosecutions were reported from 20 countries with 40% taking place in the United States. Nursing personnel comprised 86% of the healthcare providers prosecuted; physicians 12%, and 2% were allied health professionals. The number of patient deaths that resulted in a murder conviction is 317 and the number of suspicious patient deaths attributed to the 54 convicted caregivers is 2113. These numbers are disturbing and demand that systemic changes in tracking adverse patient incidents associated with presence of a specific healthcare provider be implemented. Hiring practices must shift away from preventing wrongful discharge or denial of employment lawsuits to protecting patients from employees who kill.

The transcriptional landscape of the mammalian genome.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

A reference range for endogenous gamma-hydroxybutyrate in urine by gas chromatography-mass spectrometry.

Gamma-hydroxybutyrate (GHB) has been implicated in drug-facilitated sexual assault (DFSA). The interpretation of GHB levels in biological samples collected for evidence is complicated by the natural presence of this compound in the body, and by its extremely rapid elimination after ingestion. There is a lack of agreement regarding a suitable cut-off concentration, which can reliably separate endogenous concentrations in urine from those reflecting ingestion. We have developed a method for the analysis of low levels of GHB in urine and have used it to establish a reference range for normal females. The method uses liquid-liquid extraction, silyl-derivatization, and gas chromatographic-mass spectrometric analysis. The limit of detection was 0.1 mg/L, and the method was linear from 0.1 to 5.0 mg/L. Our analysis of 50 urine samples donated by normal women indicates an upper limit of normal for urinary GHB of 1.46 mg/L or 323 microg GHB/mmol of creatinine. We propose that a 5 mg/L cut-off for urine GHB concentration, or 1000 microg GHB/mmol creatinine, will separate endogenous GHB concentrations from those reflecting GHB ingestion in antemortem samples with greater than 99% confidence, providing that a specific assay method comparable with that we describe is used. We demonstrate that urinary GHB concentrations fall with age and that this can be corrected for by measurement of the GHB/creatinine ratio.

Deaths associated with MBDB misuse.

The use of phenethylamines in the dance scene is now well established. Apart from amphetamine, the commonest phenethylamine encountered in clinical and forensic settings is 3,4-methylenedioxymethamphetamine (MDMA) commonly known as ecstasy. Other phenethylamines, which have similar effects are encountered, such as 3,4-methylenedioxyethylamphetamine (MDEA) and their use has resulted in death. We report two deaths associated with another less commonly encountered member of the group, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB), also known as Methyl-J and Eden.

Methadone deaths: a toxicological analysis.

AIMS: To perform a toxicological analysis of deaths involving methadone and to determine the fatal concentration of methadone in such deaths. METHODS: Deaths in which methadone was mentioned in the cause of death were identified. Deaths were divided into those associated with methadone only and deaths in which the cause of death was a combination of methadone and other drugs. Toxicological findings in these deaths were analysed and compared with previously published data. RESULTS: One hundred and eleven cases were analysed. In 55 cases, methadone poisoning was given as the sole cause of death. Fifty victims were adults, age range 17-51 years (median, 23), with five victims under 14 years of age. The mean methadone concentration in the adult deaths was 584 micrograms/litre (median, 435; range, 84-2700). In 56 cases, age range 15-49 years, (median, 28), death was ascribed to a combination of methadone and other drugs. The mean methadone concentration in these deaths was 576 micrograms/litre (median, 294; range, 49-2440). In 26 cases, multiple site sampling was performed. This revealed that there could be a 100% discrepancy between methadone concentrations, and other drugs, in samples collected in different sites in the same body. CONCLUSIONS: There is an overlap between quoted therapeutic methadone concentrations and methadone concentrations seen in fatalities. However, those dying from methadone poisoning might not be the same as those in a methadone programme. A degree of caution must be exercised in determining a fatal concentration because of the phenomenon of postmortem redistribution. Pathologists and toxicologists need to examine all the available postmortem findings in identifying the cause of death.

Multiple splicing variants of cdc25B regulate G2/M progression.

Progression through G2 phase into mitosis is regulated by the activation of the mitotic cyclin/cdk complexes, which are in turn activated cdc25B and cdc25C phosphatases. Here we report that alternate splicing produces at least five variants of cdc25B, although only cdc25B2 and cdc25B3 are detectable as proteins. Analysis of these two variants shows that cdc25B2 is expressed at lower levels relative to cdc25B3 in all cell lines tested, and the expression of both increased markedly during G2 and mitosis. Overexpression of the catalytically inactive version of either cdc25B variant produced a G2 arrest implicating both in regulating G2/M progression.

A method for the rapid detection of the zopiclone degradation product 2-amino-5-chloropyridine.

Zopiclone (Zimovane) is a cyclopyrrolone compound which exhibits hypnotic and sedative effects while also exhibiting anticonvulsant and muscle relaxant activities. The detection and quantification of zopiclone is difficult. It has a high molecular weight compared to most other commonly used drugs, therapeutic levels are not high, and it is unstable in nucleophilic solvents. A degradation product of zopiclone, 2-amino-5-chloropyridine (ACP) together with a method for its detection using high-performance liquid chromatography with diode array detection has been described previously. An account is presented of a simple method for the detection of ACP using gas chromatography with mass selective detection (GC/MS) which will facilitate detection of zopiclone use as part of a routine screen.