Acinetobacter infection--an emerging threat to human health

The genus Acinetobacter comprises a complex and heterogeneous group of bacteria, many of which are capable of causing a range of opportunistic, often catheter-related, infections in humans. However, Acinetobacter baumannii, as well as its close relatives belonging to genomic species 3 ("Acinetobacter pittii") and 13TU ("Acinetobacter nosocomialis"), are important nosocomial pathogens, often associated with epidemic outbreaks of infection, that are only rarely found outside of a clinical setting. These organisms are frequently pandrug-resistant and are capable of causing substantial morbidity and mortality in patients with severe underlying disease, both in the hospital and in the community. Several epidemic clonal lineages of A. baumannii have disseminated worldwide and seem to have a selective advantage over non-epidemic strains. The reasons for the success of these epidemic lineages remain to be elucidated, but could be related to the potential of these organisms to achieve very dynamic reorganization and rapid evolution of their genome, including the acquisition and expression of additional antibiotic resistance determinants, under fluctuating environmental and selective conditions.     

Effects of Smart Position Only (SPOT) tag deployment on white sharks Carcharodon carcharias in South Africa

We present 15 individual cases of sub-adult white sharks that were SPOT tagged in South Africa from 2003-2004 and have been re-sighted as recently as 2011. Our observations suggest SPOT tags can cause permanent cosmetic and structural damage to white shark dorsal fins depending on the duration of tag attachment. SPOT tags that detached within 12-24 months did not cause long term damage to the dorsal fin other than pigmentation scarring. Within 12 months of deployment, tag fouling can occur. After 24 months of deployment permanent damage to the dorsal fin occurred. A shark survived this prolonged attachment and there seems little compromise on the animal's long term survival and resultant body growth. This is the first investigation detailing the long term effects of SPOT deployment on the dorsal fin of white sharks.     

Modulation of Fas-FasL related apoptosis by PBN in the early phases of choline deficient diet-mediated hepatocarcinogenesis in rats

This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2-5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.     

Bias Assessment of General Chemistry Analytes using Commutable Samples

Harmonisation of reference intervals for routine general chemistry analytes has been a goal for many years. Analytical bias may prevent this harmonisation. To determine if analytical bias is present when comparing methods, the use of commutable samples, or samples that have the same properties as the clinical samples routinely analysed, should be used as reference samples to eliminate the possibility of matrix effect. The use of commutable samples has improved the identification of unacceptable analytical performance in the Netherlands and Spain. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has undertaken a pilot study using commutable samples in an attempt to determine not only country specific reference intervals but to make them comparable between countries. Australia and New Zealand, through the Australasian Association of Clinical Biochemists (AACB), have also undertaken an assessment of analytical bias using commutable samples and determined that of the 27 general chemistry analytes studied, 19 showed sufficiently small between method biases as to not prevent harmonisation of reference intervals. Application of evidence based approaches including the determination of analytical bias using commutable material is necessary when seeking to harmonise reference intervals.     

Immuno-spin trapping from biochemistry to medicine: Advances,challenges, and pitfalls. Focus on protein-centered radicals

Background

Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases.

Scope of the review

To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure.

Major conclusions

Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature.

General significance

The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered.This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Application of proton NMR spectroscopy in the study of lipid metabolites in a rat hepatocarcinogenesis model

Liver cancer is one of the most common cancers worldwide. Altered lipid metabolism in the liver is a key feature of developing liver nodules and tumors. Methods of analysis vary from the most sophisticated chromatography to the in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, we present a systematic method for the identification and quantitation of signature signals from lipid metabolites using 1D NMR proton spectroscopy. We assessed lipid metabolites in an epigenetic rat hepatocarcinogenesis model induced by treatment with a choline-deficient diet (CDAA, choline-deficient l-amino acid defined) over a period of 1 year, from the formation of steatosis, to the development of nodules and adenomas. A comparable choline-sufficient (CSAA) diet was used for the controls. The resonances of the methylene protons of the glycerol backbone in phospholipids were used to quantify the total concentration of such compounds. CDAA rat livers were found to have significantly higher levels of phospholipids, when compared to CSAA, throughout the entire carcinogenesis period. The tri-methyl protons of choline compounds serves to quantify total choline, and the vinyl and bis-allyl proton resonances can be used to not only quantify fatty acid concentrations but also to probe the number of double bonds in a fatty acid moiety. Early stages of carcinogenesis indicate a lower degree of double bonds in fatty acyl containing compounds in CDAA rat livers, when compared to CSAA. The results of this study are in agreement with those previously published in the literature on other rat hepatocarcinogenesis models.     

Phenyl-tert-butylnitrone induces tumor regression and decreases angiogenesis in a C6 rat glioma model

The prognosis of patients who are diagnosed with glioblastoma multiforme is very poor, due to the difficulty of an early and accurate diagnosis and the lack of currently efficient therapeutic compounds. The efficacy of phenyl-tert-butylnitrone (PBN) as a potential anti-glioma therapeutic drug was assessed by magnetic resonance (MR) imaging (T(1)/T(2)-weighted imaging) and MR angiography (time-of-flight imaging, in conjunction with a Mathematica-based program) methods by monitoring morphologic properties, growth patterns, and angiogenic behaviors of a moderately aggressive rat C6 glioma model. MR results from untreated rats showed the diffusive invasiveness of C6 gliomas, with some associated angiogenesis. PBN administration as a pretreatment was found to clearly induce a decrease in growth rate and tumor regression as well as preventing angiogenesis. This compound even had a 40% efficiency in reducing well-established tumors. MR findings rivaled those from histology and angiogenesis marker immunostaining evaluations. In this study we demonstrated the efficiency of PBN as a potential anti-glioma drug and found it to inhibit tumor cell proliferation and prevent vascular alterations in early stages of glioma progression. The MR methods that we used also proved to be particularly suitable in following the angiogenic behavior and treatment response of a potential anti-glioma agent in a rat C6 glioma model.     

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

Genetic analysis of chromosomal resistance to trimethoprim derived from clinical isolates of Escherichia coli

Chromosomal genes conferring resistance to trimethoprim were transferred from three independently isolated thy+ clinical strains of Escherichia coli to Escherichia coli K12 by using P1 transduction. Trimethoprim-resistant transductants were obtained less frequently than transductants of other chromosomal markers, suggesting that there were problems related to the expression of the trimethoprim resistance genes in E. coli K12. Mapping studies revealed that one of the resistance determinants was located at a similar position on the chromosome (1 min) to the fol-type mutations previously described in E. coli K12. The two remaining resistance determinants mapped at separate positions between 2.5 and 3 min on the chromosome. The presence of one of these determinants reduced the efficiency with which either donor or recipient cells carrying it could participate in conjugation mediated by the sex factor F and also resulted in phenotypic interaction with the azi gene. The mechanisms of trimethoprim resistance in the three clinical E. coli isolates studied were more complex and diverse than was expected from previous studies of E. coli K12 mutants.