In vivo detection of endotracheal tube biofilms in intubated critical care patients using catheter‐based optical coherence tomography

The formation of biofilms in the endotracheal tubes (ETTs) of intubated patients on mechanical ventilation is associated with a greater risk of ventilator‐associated pneumonia and death. New technologies are needed to detect and monitor ETTs in vivo for the presence of these biofilms. Longitudinal OCT imaging was performed in mechanically ventilated subjects at 24‐hour intervals until extubation to detect the formation and temporal changes of in vivo ETT biofilms. OCT‐derived attenuation coefficient images were used to differentiate between mucus and biofilm. Extubated ETTs were examined with optical and electron microscopy, and all imaging results were correlated with standard‐of‐care clinical test reports. OCT and attenuation coefficient images from four subjects were positive for ETT biofilms and were negative for two subjects. The processed and stained extubated ETTs and clinical reports confirmed the presence/absence of biofilms in all subjects. Our findings confirm that OCT can detect and differentiate between biofilm‐positive and biofilm‐negative groups (P < 10^?5). OCT image‐based features may serve as biomarkers for direct in vivo detection of ETT biofilms and help drive investigation of new management strategies to reduce the incidence of VAP.      

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Preliminary dielectric measurement and analysis protocol for determining the melting temperature and binding energy of short sequences of DNA in solution

Measurement of the real dielectric constant of bulk buffer solutions containing short sequences of DNA as a function of temperature through the DNA melting or denaturiztion transition can be used to determine melting temperatures, T(m), and to estimate the binding energy of the complimentary strands. We describe a preliminary dielectric measurement and analysis protocol to determine these parameters and its application to two known short sequences. The relative real dielectric constant for the bulk solutions was determined over the frequency range of 50 Hz-20 kHz and temperature range of <40-65 degrees C. The measurements were performed on dilute solutions and utilized low electric field strengths. Based on fits to the data by modified sigmoid functions, the melting temperatures, width of transition, and binding energy for the two sequences in solution were estimated. It was observed that the order of the transition appeared to be second order. The results were then compared against predictions of a number of models from the literature that provide theoretical estimates for the melting temperatures of known short sequences of DNA.     

Single strand DNA binding of simian virus 40 tumor antigen.

Simian virus 40 T antigen binds to both single and double strand DNA. The single and double strand DNA binding activity of crude T antigen preparations was evaluated by chromatography of the antigen on DNA-cellulose columns. Crude T antigen was retained on both native and denatured DNA-cellulos columns and was eluted from both columns under similar conditions. The interaction of highly purified T antigen with single and double strand DNA was evaluated by competition experiments using a DNA filter binding assay. These experiments showed that T antigen binds preferentially to single strand calf thymus DNA by more than an order of magnitude when compared to double strand calf thymus DNA.     

Managing familial risk in genetic testing

Increasing numbers of people are seeking genetic testing and uncovering information that directly concerns their biological relatives as well as themselves. This familial quality of genetic information raises ethical quandaries for physicians, particularly related to their duty of confidentiality. In this article, the American Medical Association's Council on Ethical and Judicial Affairs examines the informed consent process in the specific context of genetic testing, giving particular consideration to the handling of information that has consequences for biological relatives. Furthermore, it addresses the question of whether physicians' obligation to warn biological relatives ever should override the obligation to protect patient confidentiality.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.