Project description:The initial rise of fitness that occurs with increasing temperature is attributed to Arrhenius kinetics, in which rates of reaction increase exponentially with increasing temperature. Models based on Arrhenius typically assume single rate-limiting reactions over some physiological temperature range for which all the rate-limiting enzymes are in 100% active conformation. We test this assumption using data sets for microbes that have measurements of fitness (intrinsic rate of population growth) at many temperatures and over a broad temperature range and for diverse ectotherms that have measurements at fewer temperatures. When measurements are available at many temperatures, strictly Arrhenius kinetics are rejected over the physiological temperature range. However, over a narrower temperature range, we cannot reject strictly Arrhenius kinetics. The temperature range also affects estimates of the temperature dependence of fitness. These results indicate that Arrhenius kinetics only apply over a narrow range of temperatures for ectotherms, complicating attempts to identify general patterns of temperature dependence.
Project description:The regio- (and stereo-)selectivity and specific activity of cytochrome P450s are determined by the accessibility of potential sites of metabolism (SOMs) of the bound substrate relative to the heme, and the activation barrier of the regioselective oxidation reaction(s). The accessibility of potential SOMs depends on the relative binding free energy (??Gbind ) of the catalytically active substrate-binding poses, and the probability of the substrate to adopt a transition-state geometry. An established experimental method to measure activation energies of enzymatic reactions is the analysis of reaction rate constants at different temperatures and the construction of Arrhenius plots. This is a challenge for multistep P450-catalyzed processes that involve redox partners. We introduce a modified Arrhenius approach to overcome the limitations in studying P450 selectivity, which can be applied in multiproduct enzyme catalysis. Our approach gives combined information on relative activation energies, ??Gbind values, and collision entropies, yielding direct insight into the basis of selectivity in substrate conversion.
Project description:The structural assembly of synapses can be accomplished in a rapid time frame, although most nascent synapses formed during early development are not fully functional and respond poorly to presynaptic action potentials. The mechanisms that are responsible for this delay in synapse maturation are unknown. Histone deacetylases (HDACs) regulate the activity state of chromatin and repress gene expression through the removal of acetyl groups from histones. Class I HDACs, which include HDAC1 and HDAC2, are expressed in the CNS, although their specific role in neuronal function has not been studied. To delineate the contribution of HDAC1 and HDAC2 in the brain, we have used pharmacological inhibitors of HDACs and mice with conditional alleles to HDAC1 and HDAC2. We found that a decrease in the activities of both HDAC1 and HDAC2 during early synaptic development causes a robust facilitation of excitatory synapse maturation and a modest increase in synapse numbers. In contrast, in mature neurons a decrease in HDAC2 levels alone was sufficient to attenuate basal excitatory neurotransmission without a significant change in the numbers of detectable nerve terminals. Therefore, we propose that HDAC1 and HDAC2 form a developmental switch that controls synapse maturation and function acting in a manner dependent on the maturational states of neuronal networks.
Project description:Chimeric antigen receptors (CARs) are engineered molecules designed to endow a polyclonal T-cell population with the ability to recognize tumor-associated surface antigens. In their simplest form, CARs comprise a targeting moiety in the form of a single-chain variable fragment from an antibody connected to various intracellular signaling domains allowing for T-cell activation. This powerful approach combines the specificity of an antibody with the cytotoxic ability of a T cell. There has been much excitement since early phase trials of CAR-T cells targeting CD19 expressed on B-cell malignancies demonstrated remarkable efficacy in inducing long-term, stable remissions in otherwise relapsed/refractory disease. Despite these successes, we have just begun to understand the intricacies of CAR biology with efforts underway to utilize this platform in the treatment of other, previously refractory malignancies. Challenges currently include identification of viable cancer targets, management strategies for potentially severe and irreversible toxicities and overcoming the immunosuppressive nature of the tumor microenvironment. This review will focus on basic CAR structure and function, previous success and new approaches aimed at the broader application of CAR-T-cell therapy.
Project description:Direct aperture optimization (DAO) has been used to produce high dosimetric quality intensity-modulated radiotherapy (IMRT) treatment plans with fast treatment delivery by directly modeling the multileaf collimator segment shapes and weights. To improve plan quality and reduce treatment time for our in-house treatment planning system, we implemented a new DAO approach without using a global objective function (GFO). An index concept is introduced as an inverse form of back-projection used in the CT multiplicative algebraic reconstruction technique (MART). The index, introduced for IMRT optimization in this work, is analogous to the multiplicand in MART. The index is defined as the ratio of the optima over the current. It is assigned to each voxel and beamlet to optimize the fluence map. The indices for beamlets and segments are used to optimize multileaf collimator (MLC) segment shapes and segment weights, respectively. Preliminary data show that without sacrificing dosimetric quality, the implementation of the DAO reduced average IMRT treatment time from 13 min to 8 min for the prostate, and from 15 min to 9 min for the head and neck using our in-house treatment planning system PlanUNC. The DAO approach has also shown promise in optimizing rotational IMRT with burst mode in a head and neck test case.
Project description:Bronchiectasis is an increasingly recognised respiratory condition with limited therapeutic options and a complex spectrum of clinical manifestations that invariably includes chronic cough. As the primary presentation of bronchiectasis in most cases, chronic cough and its mechanistic underpinnings are of central importance but remain poorly understood in this setting. Bronchiectasis is also increasingly identified as an underlying cause of chronic cough highlighting the interrelationship between the two conditions that share overlapping clinical features. Several therapeutic approaches have illustrated positive effects on bronchiectasis-associated cough, however, more focused treatment of heterogeneous cough subtypes may yield better outcomes for patients. A current challenge is the identification of bronchiectasis and cough endophenotypes that may allow improved patient stratification and more targeted therapeutic matching of the right treatment to the right patient. Here we discuss the complex disease phenotypes of bronchiectasis and their interrelationship with cough while considering current and emerging treatment options. We discuss some key cough promoters in bronchiectasis including infection, allergy and immune dysfunction.
Project description:The famous Arrhenius equation is well suited to describing the temperature dependence of chemical reactions but has also been used for complicated biological processes. Here, we evaluate how well the simple Arrhenius equation predicts complex multi-step biological processes, using frog and fruit fly embryogenesis as two canonical models. We find that the Arrhenius equation provides a good approximation for the temperature dependence of embryogenesis, even though individual developmental intervals scale differently with temperature. At low and high temperatures, however, we observed significant departures from idealized Arrhenius Law behavior. When we model multi-step reactions of idealized chemical networks, we are unable to generate comparable deviations from linearity. In contrast, we find the two enzymes GAPDH and β-galactosidase show non-linearity in the Arrhenius plot similar to our observations of embryonic development. Thus, we find that complex embryonic development can be well approximated by the simple Arrhenius equation regardless of non-uniform developmental scaling and propose that the observed departure from this law likely results more from non-idealized individual steps rather than from the complexity of the system.
Project description:The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine.
Project description:Transporters are integral membrane proteins with central roles in the efficient movement of molecules across biological membranes. Many transporters exist as oligomers in the membrane. Depending on the individual transport protein, oligomerization can have roles in membrane trafficking, function, regulation and turnover. For example, our recent studies on UapA, a nucleobase ascorbate transporter, from Aspergillus nidulans, have revealed both that dimerization of this protein is essential for correct trafficking to the membrane and the structural basis of how one UapA protomer can affect the function of the closely associated adjacent protomer. Here, we review the roles of oligomerization in many particularly well-studied transporters and transporter families.
Project description:Tenascin-C is a large, multimodular, extracellular matrix glycoprotein that exhibits a very restricted pattern of expression but an enormously diverse range of functions. Here, we discuss the importance of deciphering the expression pattern of, and effects mediated by, different forms of this molecule in order to fully understand tenascin-C biology. We focus on both post transcriptional and post translational events such as splicing, glycosylation, assembly into a 3D matrix and proteolytic cleavage, highlighting how these modifications are key to defining tenascin-C function.