Enzymatic Photometric Assays for the Selective Detection of Halides.

Halides are substrates and products of a number of biotechnologically important enzymes like dehalogenases, halide methyltransferases, and halogenases. Therefore, the determination of halide concentrations in samples is important. The classical methods based on mercuric thiocyanate are very dangerous, produce hazardous waste, and do not discriminate between chloride, bromide, and iodide. In this chapter, we describe a detailed protocol for the determination of halide concentrations based on the haloperoxidase-catalyzed oxidation of halides. The resulting hypohalous acids are detected using commercially available colorimetric or fluorimetric probes. The biocatalytic nature of the assays allows them to be implemented in one-pot cascade reactions with halide-generating enzymes. Since chloride is ubiquitous in biological systems, we also describe convenient photometric assays for the selective detection of bromide and iodide in the presence of chloride, obviating the need for laborious dialyses to obtain halide-free enzymes and reagents.

Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs.

Biocatalytic alkylations are important reactions to obtain chemo-, regio- and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4-dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation.

Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium-dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 µM for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well-established GC-MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide-producing enzymes.

Luteal serum BDNF and HSP70 levels in women with premenstrual dysphoric disorder.

Premenstrual dysphoric disorder (PMDD) is a severe form of premenstrual syndrome characterized by psychological and somatic symptoms commencing in the luteal phase of the menstrual cycle and concludes with menstrual bleeding. PMDD affects 3-8 % of premenopausal women and represents a significant public health problem especially in young women. Decreased brain-derived neurotrophic factor (BDNF) levels are associated with several mental disorders. Heat-shock protein-70 (HSP70) is an important member of the molecular chaperone system, which provides a molecular defense against proteotoxic stress. We hypothesized that there would be changed levels of BDNF and HSP70 in women with PMDD compared with non-symptomatic women, reflecting impaired and/or activated stress-related responses involved in the underlying pathogenesis of PMDD. Female medical students were screened, and 24 women without premenstrual symptoms and 25 women with PMDD were enrolled in the study. Psychiatric evaluation and the Daily Record of Severity of Problems-Short Form were used for two consecutive menstrual cycles to diagnose PMDD. Serum BDNF and HSP70 levels were assessed in the third luteal phase. Participants with PMDD had significantly higher serum BDNF and HSP70 levels compared with controls, and there was a significant positive correlation between serum BDNF and HSP70 levels. Increased HSP70 levels may reflect cellular distress in PMDD. Increased serum BDNF levels in the luteal phase in subjects with PMDD may reflect a compensation process, which results in subsequent improvement of PMDD-associated depressive symptoms in the follicular phase. Thus, increased serum BDNF levels may be indicative of a compensating capacity in PMDD.

Blood cultures positive for coagulase-negative staphylococci: antisepsis, pseudobacteremia, and therapy of patients.

A blood culture cohort study investigating issues related to isolation of coagulase-negative staphylococci (CoNS) and other skin microflora is reported. Data were collected over 12 weeks to determine the incidence of significant CoNS bacteremia versus that of pseudobacteremia (contaminants) and to evaluate drug therapy in patients with cultures positive for CoNS. In addition, the effectiveness of 0.2% chlorine peroxide as a bactericidal disinfectant was compared to that of 10% providone iodine. A total of 3,276 cultures of blood from 1,433 patients were evaluated in the study. Eighty-nine cultures were positive for skin flora, with 81 of 89 (91%) involving CoNS. The incidence of significant CoNS bacteremia was 20 of 81 (24.7%), that of indeterminate bacteremia was 10 of 81 (12.3%), and that of contamination was 59 of 81 (72.8%). The incidence of significant bacteremia involving CoNS was double the 10 to 12% rate based on previous estimations at our institutions. In tests with the two bactericidal disinfectants, 22 of 1,639 cultures (1.3%) in the chlorine peroxide group versus 37 of 1,637 (2.3%) in the providone iodine group were considered contaminated (P = 0.065). Rates of contamination for venipuncture versus catheter collection were not significantly different (P = 0.46). The overall contamination rate was 59 of 3,276 (1.8%), which is consistent with the lower end of published quality assurance benchmark standards. The low rate was believed to be due to the professional phlebotomy staff in our institutions. There was excellent agreement between retrospective analysis by reviewers, when formal criteria were used, and the attending physicians' intuitive clinical impressions in the classification of significant bloodstream infections (100% agreement) or contamination (95% agreement). However, physicians still used antimicrobial agents to treat nearly one-half of the patients with contaminated blood cultures, with vancomycin being misused in 34% of patients. In addition, 10% of patients with significant bacteremia were treated with inappropriate agents. There were no significant adverse events or prolonged hospital stays due to the unnecessary use of vancomycin; however, the additional costs of treating patients whose cultures contained CoNS contaminants was estimated to be $1,000 per patient. Measures to limit the unnecessary use of vancomycin (and other agents) are important.

Limb gangrene secondary to thromboembolic disease of the newborn.

Thromboembolic disease of the newborn is a well-known entity to neonatologists. When severe arterial compromise threatens limb survival, the orthopaedic surgeon may be called on to assist in the management of this disorder. A case of spontaneous postnatal ischemic gangrene of an upper extremity that resulted in amputation in a premature infant is presented; management options are discussed. Newborns are prone to thromboembolism due to a fibrinolytic capability that is significantly different from that of adults.

Femoral neck fractures in young adults.

In 17 patients with femoral neck fractures who were between 15 and 40 years old the incidence of aseptic necrosis in patients followed more than 2 years was 18.7 per cent. The fracture was associated with other severe injuries in 40 per cent of the cases, probably reflecting the level of violence necessary to induce the fracture in young adults. Two of the 7 fractures treated with large-bore internal fixatives required secondary procedures to correct fracture displacement (this was not a complication with threaded devices). While the incidence of aseptic necrosis is no higher than that in other adult series, subsequent degenerative changes may supervene in many femoral neck fractures in this age group.