Enhancement of microbicidal efficacy of chemical disinfectants when combined with ultrasound technology.

AIMS: This study investigated the antimicrobial efficacy of ultrasound technology (US) in combination with two different disinfectants (Disinfectant A and Disinfectant B), containing peracetic acid (PAA) and quaternary ammonium compounds (QACs), respectively, against two sporigenic pathogens, Aspergillus brasiliensis and Bacillus subtilis. METHODS AND RESULTS: The microbicidal activity of the coupled treatment was compared with the use of the disinfectants alone, and the efficacy of the disinfection strategies was evaluated by the log reduction of the population of the microorganism inoculated onto stainless-steel surface. The combination treatment resulted in a log reduction of 5.40 and 3.88 (Disinfectant A + US) against A. brasiliensis and B. subtilis, at 850 and 500 ppm PAA, compared to 265 and 122 (Disinfectant A only). For Disinfectant B, in combination with US, showed a logarithmic reduction of 5.04 and 4.79 against A. brasiliensis and B. subtilis at 078% v v-1 and 392% v v-1 QACs, respectively, vs. 1.58 and 1.64 (Disinfectant B only). Moreover, no colonies or not statistically significant growth was observed within the US bath containing the disinfectant. CONCLUSIONS: The antimicrobial efficacy of the two disinfectants was greatly enhanced when used in combination with US, and this also makes it possible to avoid the overuse of chemicals for disinfection.

Mycobacterium hassiacum: a thermophilic Mycobacterium species to demonstrate thermal disinfection of medical devices.

OBJECTIVE: Reprocessing reusable medical devices is crucial in the healthcare industry. To ensure patient safety, strict standards are dictated to validate thermal disinfection in automated washer-disinfectors. The United States Food and Drug Administration (FDA) has specific recommendations on the vegetative bacterial challenge but comparatively vague guidance on the use of a thermophilic Mycobacterium strain for thermal disinfection studies. This study aims to compare thermal resistance of Mycobacterium hassiacum and Mycobacterium terrae and determine which strain is suitable for medical device thermal disinfection validation testing in automated washer-disinfectors. RESULTS: Thermal resistance was demonstrated in vitro by calculating D-values for each strain at different exposure temperatures, and correlated with actual in situ processing conditions. M. terrae was completely killed (> 7 log reduction) at temperatures above 68 °C, with D-values between 46.6 and 27.8 s at temperatures between 59.5 and 67.2 °C. M. hassiacum was completely killed (> 8 log reduction) at temperatures above 75 °C, with D-values between 82.1 and 21.7 s at temperatures ranging between 69.2 and 73.6 °C. In vitro results were correlated in a washer-disinfector performance validation setup.

Stabilization of cellular RNA in blood during storage at room temperature: a comparison of cell-free RNA BCT(®) with K3EDTA tubes.

BACKGROUND: Messenger RNA (mRNA) expression levels in blood cells are important in disease diagnosis, prognosis and biomarker discovery research. Accurate measurements of intracellular mRNA levels in blood cells depend upon several pre-analytical factors, including delays in RNA extraction from blood after phlebotomy. Dramatic changes in mRNA expression levels caused by delays in blood sample processing may render such samples unsuitable for gene expression analysis. OBJECTIVES: This study was conducted to evaluate a blood collection tube, cell-free RNA-BCT(®) (RNA-BCT), for its ability to stabilize mRNA expression level in blood cells post-phlebotomy using indicator mRNAs in reverse transcription quantitative real-time PCR (RT-qPCR) assays. METHODS: Blood samples from presumed healthy donors were drawn into both RNA-BCT and K3EDTA tubes and maintained at room temperature (18-22°C). The samples were processed to obtain white blood cells (WBCs) at days 0, 1, 2 and 3. Total cellular RNA was extracted from WBCs and mRNA concentrations were quantified by RT-qPCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), c-fos, and p53 transcripts. RESULTS: While blood cells isolated from K3EDTA tubes showed significant changes in cellular mRNA concentrations for GAPDH, c-fos, and p53, these mRNAs concentrations were stable in blood drawn into RNA-BCT. CONCLUSION: The reagent in the RNA-BCT device stabilizes cellular mRNA concentrations for GAPDH, c-fos and p53 for at least three days at room temperature.

Stabilization of circulating tumor cells in blood using a collection device with a preservative reagent.

BACKGROUND: The enumeration and characterization of circulating tumor cells (CTCs) in the blood of cancer patients is useful for cancer prognostic and treatment monitoring purposes. The number of CTCs present in patient blood is very low; thus, robust technologies have been developed to enumerate and characterize CTCs in patient blood samples. One of the challenges to the clinical utility of CTCs is their inherent fragility, which makes these cells very unstable during transportation and storage of blood samples. In this study we investigated Cell-Free DNA BCT™ (BCT), a blood collection device, which stabilizes blood cells in a blood sample at room temperature (RT) for its ability to stabilize CTCs at RT for an extended period of time. METHODS: Blood was drawn from each donor into K3EDTA tube, CellSave tube and BCT. Samples were then spiked with breast cancer cells (MCF-7), transported and stored at RT. Spiked cancer cells were counted using the Veridex CellSearch™ system on days 1 and 4. The effect of storage on the stability of proteins and nucleic acids in the spiked cells isolated from K3EDTA tube and BCT was determined using fluorescence staining and confocal laser scanning microscopy. RESULTS: MCF-7 cell recovery significantly dropped when transported and stored in K3EDTA tubes. However, in blood collected into CellSave tubes and BCTs, the MCF-7 cell count was stable up to 4 days at RT. Epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) in MCF-7 cells isolated from BCTs was stable at RT for up to 4 days, whereas in MCF-7 cells isolated from K3EDTA blood showed reduced EpCAM and CK protein expression. Similarly, BCTs stabilized c-fos and cyclin D1 mRNAs as compared to K3EDTA tubes. CONCLUSION: Cell-Free DNA™ BCT blood collection device preserves and stabilizes CTCs in blood samples for at least 4 days at RT. This technology may facilitate the development of new non-invasive diagnostic and prognostic methodologies for CTC enumeration as well as characterization.

Mdm2 binds to Nbs1 at sites of DNA damage and regulates double strand break repair.

Mdm2 directly regulates the p53 tumor suppressor. However, Mdm2 also has p53-independent activities, and the pathways that mediate these functions are unresolved. Here we report the identification of a specific association of Mdm2 with Mre11, Nbs1, and Rad50, a DNA double strand break repair complex. Mdm2 bound to the Mre11-Nbs1-Rad50 complex in primary cells and in cells containing inactivated p53 or p14/p19ARF, a regulator of Mdm2. Further analysis revealed that Mdm2 directly bound to Nbs1 but not to Mre11 or Rad50. Amino acids 198-314 of Mdm2 were required for Mdm2/Nbs1 association, and neither the N terminus forkhead-associated and breast cancer C-terminal domains nor the C terminus Mre11 binding domain of Nbs1 mediated the interaction of Nbs1 with Mdm2. Mdm2 co-localized with Nbs1 to sites of DNA damage following gamma-irradiation. Notably, Mdm2 overexpression inhibited DNA double strand break repair, and this was independent of p53 and ARF, the alternative reading frame of the Ink4alocus. The delay in DNA repair imposed by Mdm2 required the Nbs1 binding domain of Mdm2, but the ubiquitin ligase domain in Mdm2 was dispensable. Therefore, Nbs1 is a novel p53-independent Mdm2 binding protein and links Mdm2 to the Mre11-Nbs1-Rad50-regulated DNA repair response.

Loss of one allele of ARF rescues Mdm2 haploinsufficiency effects on apoptosis and lymphoma development.

The tumor suppressor p19ARF inhibits Mdm2, which restricts the activity of p53. Complicated feedback and control mechanisms regulate ARF, Mdm2, and p53 interactions. Here we report that ARF haploinsufficiency completely rescued the p53-dependent effects of Mdm2 haploinsufficiency on B-cell development, survival, and transformation. In contrast to Mdm2+/- B cells, Mdm2+/- B cells deficient in ARF were similar to wild-type B cells in their rates of growth and apoptosis and activation of p53. Consequently, the profoundly reduced numbers of B cells in Mdm2+/-Emu-myc transgenic mice were restored to normal levels in ARF+/-Mdm2+/-Emu-myc transgenics. Additionally, ARF+/-Mdm2+/-Emu-myc transgenics developed lymphomas at rates analogous to those observed for wild-type Emu-myc transgenics, demonstrating that loss of one allele of ARF rescued the protracted lymphoma latency in Mdm2+/-Emu-myc transgenics. Importantly, in ARF+/-Mdm2+/-Emu-myc transgenic lymphomas, p53 was inactivated at the frequency observed in lymphomas of wild-type Emu-myc transgenics. Collectively, these results support a model whereby the stoichiometry of Mdm2 and ARF controls apoptosis and tumor development, which should have significant implications in the treatment of malignancies that have inactivated ARF.

Mdm2 haplo-insufficiency profoundly inhibits Myc-induced lymphomagenesis.

Mdm2 harnesses the p53 tumor suppressor, yet loss of one Mdm2 allele in Mdm2(+/-) mice has heretofore not been shown to impair tumor development. Here we report that Mdm2 haplo-insufficiency profoundly suppresses lymphomagenesis in E micro -myc transgenic mice. Mdm2(+/-)E micro -myc transgenics had greatly protracted rates of B cell lymphoma development with life spans twice that of wild-type transgenic littermates. Im paired lymphoma development was associated with drastic reductions in peripheral B cell numbers in Mdm2(+/-)E micro -myc transgenics, and primary pre-B cells from Mdm2(+/-)E micro -myc transgenics and Mdm2(+/-) littermates were extremely susceptible to spontaneous apoptosis. Loss of p53 rescued all of the effects of Mdm2 haplo-insufficiency, indicating they were p53 dependent. Furthermore, half of the lymphomas that ultimately emerged in Mdm2(+/-)E micro -myc transgenics harbored inactivating mutations in p53, and the majority overcame haplo-insufficiency by overexpressing Mdm2. These results support the concept that Mdm2 functions are rate limiting in lymphomagenesis and that targeting Mdm2 will enhance p53-mediated apoptosis, compromising tumor development and/or maintenance.

p21(Cip1) Promotes cyclin D1 nuclear accumulation via direct inhibition of nuclear export.

There is increasing evidence that p21(Cip1) and p27(Kip1) are requisite positive regulators of cyclin D1.CDK4 assembly and nuclear accumulation. Both Cip and Kip proteins can promote nuclear accumulation of cyclin D1, but the underlying mechanism has not been elucidated. We now provide evidence that p21(Cip1) promotes the nuclear accumulation of cyclin D1 complexes via inhibition of cyclin D1 nuclear export. In vivo, we demonstrate that p21(Cip1) can inhibit glycogen synthase kinase 3 beta-triggered cyclin D1 nuclear export and phosphorylation-dependent nucleocytoplasmic shuttling. Furthermore, we find that cyclin D1 nuclear accumulation in p21/p27 null cells can be restored through inhibition of CRM1-dependent nuclear export. The ability of p21(Cip1) to inhibit cyclin D1 nuclear export correlates with its ability to bind to Thr-286-phosphorylated cyclin D1 and thereby prevents cyclin D1.CRM1 association.