Implications of paper vs stainless steel biological indicator substrates for formaldehyde gas decontamination

Aims: This study was undertaken to determine the effectiveness of biological indicators currently being employed during formaldehyde decontamination. Data suggest that detectable amounts of formaldehyde are absorbed into the paper strips contained in currently used biological indicators. Absorbed formaldehyde has the potential to inhibit the growth of indicator spores, thus leading to false negative results. Indicators composed of either stainless steel carriers or paper strips were investigated to determine whether stainless steel carriers can be used as an alternative to paper strip indicators. Methods and Results: Biological indicators were exposed to formaldehyde gas and were tested for the presence of formaldehyde and any possible inhibition of spore growth. Absorbed formaldehyde was detected in the paper strip carriers while no formaldehyde was detected from any of the stainless steel carriers. Exposed paper strips were found to inhibit growth of up to 1 × 10⁶ spores while the stainless steel carriers did not inhibit the growth of spores. Conclusions: During decontamination, biological indicators composed of paper spore strips absorb formaldehyde and inhibit growth of any surviving spores. Stainless steel carriers do not absorb formaldehyde and are an ideal alternative substrate for biological indicators. Significance and Impact of the Study: The popular paper strip biological indicator can lead to false negative results during decontamination and is unsuitable for validating formaldehyde decontamination.     

Expression and localization of CLC chloride transport proteins in the avian retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl(-)/H(+) antiporters, ClCs 3-7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl(-) can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.     

A systems-based mathematical modelling framework for investigating the effect of drugs on solid tumours

Background and Purpose

Most information on the dose-response of radiation-induced cancer is derived from data on the A-bomb survivors. Since, for radiation protection purposes, the dose span of main interest is between zero and one Gy, the analysis of the A-bomb survivors is usually focused on this range. However, estimates of cancer risk for doses larger than one Gy are becoming more important for radiotherapy patients. Therefore in this work, emphasis is placed on doses relevant for radiotherapy with respect to radiation induced solid cancer.

Materials and methods

For various organs and tissues the analysis of cancer induction was extended by an attempted combination of the linear-no-threshold model from the A-bomb survivors in the low dose range and the cancer risk data of patients receiving radiotherapy for Hodgkin's disease in the high dose range. The data were fitted using organ equivalent dose (OED) calculated for a group of different dose-response models including a linear model, a model including fractionation, a bell-shaped model and a plateau-dose-response relationship.

Results

The quality of the applied fits shows that the linear model fits best colon, cervix and skin. All other organs are best fitted by the model including fractionation indicating that the repopulation/repair ability of tissue is neither 0 nor 100% but somewhere in between. Bone and soft tissue sarcoma were fitted well by all the models. In the low dose range beyond 1 Gy sarcoma risk is negligible. For increasing dose, sarcoma risk increases rapidly and reaches a plateau at around 30 Gy.

Conclusions

In this work OED for various organs was calculated for a linear, a bell-shaped, a plateau and a mixture between a bell-shaped and plateau dose-response relationship for typical treatment plans of Hodgkin's disease patients. The model parameters (α and R) were obtained by a fit of the dose-response relationships to these OED data and to the A-bomb survivors. For any three-dimensional inhomogenous dose distribution, cancer risk can be compared by computing OED using the coefficients obtained in this work.     

siRNA-like double-stranded RNAs are specifically protected against degradation in human cell extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time.     

Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus

Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.     

Protection of DNA and membrane from gamma radiation induced damage by gallic acid

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a naturally occurring plant phenol. In vitro and in vivo studies have shown that this phytochemical protected DNA and membranes against ionizing radiation. Rat liver microsomes and plasmid pBR322 DNA were exposed to various doses of gamma radiation in presence and absence of GA. Exposure of the microsomes to gamma radiation resulted in the formation of peroxides of membrane lipids measured as thiobarbituric acid reactive substances and presence of GA during irradiation prevented the formation of lipid peroxidation. Gamma irradiation of plasmid DNA resulted in induction of strand breaks in DNA resulting in disappearance of the supercoiled (ccc) form. Presence of GA during irradiation protected the DNA from undergoing the strand breaks. In in vivo studies it was found that whole body exposure of mice to gamma radiation (4 Gy) increased the formation of lipid peroxides in various tissues and damage to cellular DNA (as measured by alkaline comet assay) in peripheral blood leucocytes. Administration of GA to mice prior to whole body radiation exposure reduced the peroxidation of lipids and the damage to the cellular DNA indicating in vivo radiation protection of membranes and DNA by GA. (Mol Cell Biochem 278: 111–117, 2005)