Biomedical subjects

Mark O'Malley

Publications and source records attributed to Mark O'Malley.

4 recordsLinked to original sources

PubMed2004

Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness.

BACKGROUND: The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency. METHODS: To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A. RESULTS: In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (P(hCMV*-1)) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original P(hCMV*-1) in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses.

Animals↗

PubMed2003

Automated processing of the single-lead electrocardiogram for the detection of obstructive sleep apnoea.

A method for the automatic processing of the electrocardiogram (ECG) for the detection of obstructive apnoea is presented. The method screens nighttime single-lead ECG recordings for the presence of major sleep apnoea and provides a minute-by-minute analysis of disordered breathing. A large independently validated database of 70 ECG recordings acquired from normal subjects and subjects with obstructive and mixed sleep apnoea, each of approximately eight hours in duration, was used throughout the study. Thirty-five of these recordings were used for training and 35 retained for independent testing. A wide variety of features based on heartbeat intervals and an ECG-derived respiratory signal were considered. Classifiers based on linear and quadratic discriminants were compared. Feature selection and regularization of classifier parameters were used to optimize classifier performance. Results show that the normal recordings could be separated from the apnoea recordings with a 100% success rate and a minute-by-minute classification accuracy of over 90% is achievable.

Adult↗

PubMed2002

The type 1 interleukin-1 receptor is essential for the efficient activation of microglia and the induction of multiple proinflammatory mediators in response to brain injury.

Interleukin-1 (IL-1) is induced immediately after insults to the brain, and elevated levels of IL-1 have been strongly implicated in the neurodegeneration that accompanies stroke, Alzheimer's disease, and multiple sclerosis. In animal models, antagonizing IL-1 has been shown to reduce cell death; however, the basis for this protection has not been elucidated. Here we analyzed the response to penetrating brain injury in mice lacking the type 1 IL-1 receptor (IL-1R1) to determine which cellular and molecular mediators of tissue damage require IL-1 signaling. At the cellular level, fewer amoeboid microglia/macrophages appeared adjacent to the injured brain tissue in IL-1R1 null mice, and those microglia present at early postinjury intervals retained their resting morphology. Astrogliosis also was mildly abrogated. At the molecular level, cyclooxygenase-2 (Cox-2) and IL-6 expression were depressed and delayed. Interestingly, basal levels of Cox-2, IL-1, and IL-6 were significantly lower in the IL-1R1 null mice. In addition, stimulation of vascular cell adhesion molecule-1 mRNA was depressed in the IL-1R1 null mice, and correspondingly, there was reduced diapedesis of peripheral macrophages in the IL-1R1 null brain after injury. This observation correlated with a reduced number of Cox-2+ amoeboid phagocytes adjacent to the injury. In contrast, several molecular aspects of the injury response were normal, including expression of tumor necrosis factor-alpha and the production of nerve growth factor. Because antagonizing IL-1 protects neural cells in experimental models of stroke and multiple sclerosis, our data suggest that cell preservation is achieved by abrogating microglial/macrophage activation and the subsequent self-propagating cycle of inflammation.

Animals↗

PubMed2002

Agonist-antagonist common drive during fatiguing knee extension efforts using surface electromyography.

AIM: This study examined the electromyographic (EMG) activity of knee extensor agonists and a knee extensor antagonist muscle during fatiguing isometric extensions across a range of force levels. METHODS: Five female subjects performed isometric knee extensions at 25%, 50%, 75% and 100% of their maximal voluntary contraction (MVC) with the knee flexed to 75 degrees. Surface EMG (SEMG) was recorded with bipolar electrodes from the vastus lateralis (VL), vastus medialis (VM), rectus femoris (RF) and biceps femoris (BF) and the root-mean-squared (RMS) amplitude and the percentage frequency compression of these recordings were calculated. Commonality and cross talk between recordings were also examined. RESULTS: Cross talk between recordings was deemed negligible despite significant levels of commonality between the agonist and antagonist SEMG, which was attributed to common drive. SEMG RMS amplitude increased significantly for all muscles during the 25%, 50%, 75% MVC knee extensions until task failure, and decreased significantly for 100% MVC. The frequency spectrum of the SEMG compressed significantly for all muscles and % MVC levels. The VM, VL and BF SEMG recordings responded similarly to fatigue. The RF's frequency spectrum compressed to a significantly higher degree. CONCLUSIONS: The VM, VL, RF, and BF fatigue in parallel, with high similarity between VM, VL and BF, giving support to the concept of a shared agonist-antagonist motoneuron pool.

Adult↗