Cancer‐associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse‐strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non‐epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal‐binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3′ end of the 5p miRNA strand of a pre‐mRNA hairpin. To investigate the effects of these cancer‐associated 'hotspot' mutations, we engineered mouse DICER1‐deficient ES cells to express wild‐type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal‐binding site mutations were compared to each other and to wild‐type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation‐carrying cells were distinct from both wild‐type and DICER1‐deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p‐derived miRNAs. We therefore conclude that cancer‐associated somatic hotspot mutations of DICER1, affecting any one of four metal‐binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Technique for axillary radiotherapy using computer‐assisted planning for high‐risk skin cancer†

High‐risk skin cancer arising on the upper limb or trunk can cause axillary nodal metastases. Previous studies have shown that axillary radiotherapy improves regional control. There is little published work on technique. Technique standardization is important in quality assurance and comparison of results especially for trials. Our technique, planned with CT assistance, is presented. To assess efficacy, an audit of patients treated in our institution over a 15‐month period was conducted. Of 24 patients treated, 13 were treated with radical intent, 11 with this technique. With a follow up of over 2 years, the technique had more than a 90% (10/11) regional control in this radical group. Both of the radical patients who were not treated according to the technique had regional failure. One case of late toxicity was found, of asymptomatic lymphoedema in a radically treated patient. This technique for axillary radiotherapy for regional control of skin cancer is acceptable in terms of disease control and toxicity as validated by audit at 2 years.     

Fate of the anthelmintic,phenothiazine, in man

1. Radiolabelled [(35)S]-phenothiazine has been administered orally to two healthy adult male volunteers (6 mg kg(-1) body weight). Faeces were the major route of excretion of radioactivity (68%), the remainder being eliminated via the urine (32%) with an estimated urinary half-life (biphasic) of 6-16 h. Over the 5 days of the study a complete recovery of radioactivity was achieved. 2. From urinary data, it was shown that metabolism occurred via ring carbon oxidation to form phenothiazone and thionol and via ring sulphur oxidation to form phenothiazine sulphoxide. The majority of urinary material (92%) was present in the form of conjugates of phenothiazine and phenothiazone. Only unchanged phenothiazine was detected in the faeces. Phenothiazine sulphoxide was reduced to phenothiazine during incubation with faecal homogenates.     

Pneumomediastinum, an unusual complication of facial trauma

Pneumomediastinum is often an incidental finding following a blunt or penetrating trauma to the neck or chest. We report a rare case of pneumomediastinum following an isolated facial trauma that was diagnosed on imaging. We also review the clinical signs of this condition, its radiologic characteristics, and the 18 previously reported cases of pneumomediastinum following facial trauma.     

An alternative use of the surgical mask for the protection of healthcare workers managing epistaxis

Epistaxis has a reported prevalence of 10-12 per cent in the general population and is thus one of the commonest emergencies in Otolaryngology. We suggest that the patient uses a surgical mask to prevent blood spray during epistaxis management. Utilizing the described technique we have found that protective clothing worn by the healthcare professional is consistently 'blood-free' at the end of the procedure. This is in contrast to findings when no facemask is applied.     

Phenylalanine 4-monooxygenase and the S-oxidation of S-carboxymethyl-L-cysteine

The identity of the enzyme responsible for the S-oxidation of the mucolytic S-substituted L-cysteine drug, S-carboxymethyl-L-cysteine (SCMC), has been actively investigated for the last 10 years. A genetic polymorphism exists in the oxidation of the thioether moiety that has been identified as a disease susceptibility factor in a number of degenerative diseases. This polymorphism has also been implicated in the wide variation in clinical response to SCMC therapy in man. To date little is known about the molecular enzymology of this reaction but a previous investigation revealed that rat activated phenylalanine 4-monooxygenase (PAH) could S-oxidise both Met- and S-methyl-L-cysteine (SMC) to their S-oxide metabolites. We have investigated the hypothesis that SCMC was also a substrate for activated PAH in the cytosolic faction of the Wistar rat. 1. Substrate and inhibitor investigation revealed that SCMC was a substrate for activated PAH activity in vitro. 2. The large aromatic amino acid hydroxylase monoclonal antibody and the Fe3+ chelator, deferoxamine, completely inhibited both Phe and SCMC oxidation to their respective metabolites. 3. Analysis of the Dixon plots revealed that both Phe and SCMC competitively inhibited each other's oxidation. 4. Correlation studies showed that the rate of production of Tyr was positively correlated to the production of both SCMC and SMC S-oxides in 20 female Wistar rat hepatic cytosolic fractions. These results strongly support the hypothesis that PAH is the enzyme responsible for SCMC S-oxidation in the rat.     

The effect of cysteine analogues on the excretion of urinary sulphate in the rat following cysteine administration

A major pathway for the production of sulphate within the mammalian body is known to be via the oxidative degradation of the sulphur moiety within the amino acid, L-cysteine. The ability of two structurally similar sulphur-containing drugs, the anti-rheumatic agent, D-penicillamine, and the mucoactive compound, S-carboxymethyl-L-cysteine, to interfere with this sulphate production was investigated. Co-administration to the male rat of D-penicillamine (p.o.) and S-carboxymethyl-L-cysteine (p.o.) with [35S]-L-cysteine (i.p.) led to a significant decrease in the subsequent urinary elimination of inorganic sulphate whilst having no measurable effect on organic sulphate excretion. The co-administration of L-valine, an amino acid not containing sulphur, had no effect. It is not known where, within the complex sequence of events surrounding the degradation of cysteine to sulphate, that D-penicillamine or S-carboxymethyl-L-cysteine may interact.