Both Ligand- and Cell-Specific Parameters Control Ligand Agonism in a Kinetic Model of G Protein–Coupled Receptor Signaling

G protein–coupled receptors (GPCRs) exist in multiple dynamic states (e.g., ligand-bound, inactive, G protein–coupled) that influence G protein activation and ultimately response generation. In quantitative models of GPCR signaling that incorporate these varied states, parameter values are often uncharacterized or varied over large ranges, making identification of important parameters and signaling outcomes difficult to intuit. Here we identify the ligand- and cell-specific parameters that are important determinants of cell-response behavior in a dynamic model of GPCR signaling using parameter variation and sensitivity analysis. The character of response (i.e., positive/neutral/inverse agonism) is, not surprisingly, significantly influenced by a ligand's ability to bias the receptor into an active conformation. We also find that several cell-specific parameters, including the ratio of active to inactive receptor species, the rate constant for G protein activation, and expression levels of receptors and G proteins also dramatically influence agonism. Expressing either receptor or G protein in numbers several fold above or below endogenous levels may result in system behavior inconsistent with that measured in endogenous systems. Finally, small variations in cell-specific parameters identified by sensitivity analysis as significant determinants of response behavior are found to change ligand-induced responses from positive to negative, a phenomenon termed protean agonism. Our findings offer an explanation for protean agonism reported in β2-adrenergic and α2A-adrenergic receptor systems

Heart Rate and Blood Flow Velocity Variability in the Human Fetus

Much of what we know about the embryonic circulation Is derived from studies of the chick embryo (Clark and Hu 1982). The similarities between the chick, rat (Nakazawa 1988) and fetal lamb (Kirkpatrick 1976) suggest that, while the details of functional change may vary, common mechanisms are expressed In these animal groups (Nakazawa 1988). Some of the mechanisms that control the cardiovascular system in the mature animal are expressed early In development (Clark 1990). The primary determinants of cardiovascular function in the embryo as in the mature animal are preload, afterload, heart rate and myocardial contractility. These factors regulate cardiac output before the development of the functioning autonomic nervous system. The Frank-Starling relationship Is operative and effective in both the fetal lamb heart (Kirkpatrick 1976) and the chick embryo (Wagman 1990). After maturation of the autonomic nervous system, both the parasympathetic and sympathetic systems control cardiovascular function in the fetal lamb (Nuwayhid 1975)

Emerging treatment options for BRAF-mutant colorectal cancer.

The personalization of cancer care is rooted in the premise that there are subsets of patients with tumors harboring clinically relevant targets for patient-specific treatments. Colorectal cancer (CRC) is a disease that has historically been notable for its dearth of biomarkers that are predictive of response to targeted therapies. In recent years, BRAFV600E-mutated CRC has emerged as a distinct biologic entity, typically refractory to standard chemotherapy regimens approved for the treatment of metastatic CRC and associated with a dismal prognosis. Multiple clinical trials sought to replicate the successes of targeted therapies seen in BRAFV600E-mutated melanoma without success; metastatic BRAFV600E-mutated CRC is clearly a distinct biologic entity. We review a number of recent studies demonstrating the evidence of modest responses to combinations of BRAF, EGFR, and/or MEK inhibition in patients with metastatic BRAFV600E-mutated CRC; however, despite advances, overall survival remains far inferior for these patients compared to their BRAF-wild-type counterparts. Development of combination therapies to impede signaling through the MAPK pathway through alternate targets remains an area of active investigation. Reflecting the rapid evolution of efforts for this small subset of CRC patients, the first-ever Phase III study is now underway evaluating the combination of BRAF, EGFR, and MEK inhibition. Immunotherapies are also an area of active research, particularly for the subset of patients with tumors that are also microsatellite instability (MSI) high. Here, we summarize the current landscape and emerging data on the molecular, clinical, and therapeutic aspects of BRAF-mutant CRC

Analysis and modeling of control tasks in dynamic systems

Copyright © 2002 IEEEMost applications of evolutionary algorithms deal with static optimization problems. However, in recent years, there has been a growing interest in time-varying (dynamic) problems, which are typically found in real-world scenarios. One major challenge in this field is the design of realistic test-case generators (TCGs), which requires a systematic analysis of dynamic optimization tasks. So far, only a few TCGs have been suggested. Our investigation leads to the conclusion that these TCGs are not capable of generating realistic dynamic benchmark tests. The result of our research is the design of a new TCG capable of producing realistic nonstationary landscapesRasmus K. Ursem, Thiemo Krink, Mikkel T. Jensen, and Zbigniew Michalewic

Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications

Calmodulin (CaM) is a widely studied Ca^(2+)-binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca^(2+) or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications

Assessment of fetal heart rate variability and velocity variability by Doppler velocimetry of the descending aorta at 10-20 weeks of gestation

Objectives: Determination of gestational age-related modulations in fetal heart rate and descending aorta blood flow velocity in the early human fetus and comparison of aortic variability data with data obtained from the umbilical artery. It is hypothesized that these modulations present in the umbilical artery also occur in the descending aorta. Methods: Doppler studies of descending aorta velocity waveforms were performed at 10-20 weeks in 55 normal pregnant women. In 24 of the 55 women, Doppler recordings from both the descending aorta and the umbilical artery were collected. Absolute values and variability of fetal heart rate, peak systolic and time-averaged velocities were determined from flow velocity waveforms of at least 18 s in duration. Results: From 10 to 20 weeks of gestation, the descending aorta peak systolic and time-averaged velocities increased, whereas the fetal heart rate decreased. The descending aorta peak systolic variability also increased. However, the time-averaged velocity variability and the fetal heart rate variability remained constant during the study period. In the subset of 24 women, the fetal heart rate variability and velocity variability data from the descending aorta and umbilical artery were not significantly different. Conclusions: Reproducible fetal heart rate and velocity variability data can be derived from the descending aorta and umblilical artery. The increase in heart rate variability observed in the umbilical artery was not seen in recordings obtained from the descending aorta. Different fetal activity states may be the underlying mechanism for these heart rate variability discrepancies

Athymic nude rat. III natural cell mediated cytotoxicity.

Homozygous rnu/rnu and heterozygous +/rnu rats were investigated and compared with each other for the existence of natural cell-mediated cytotoxicity. Investigated were total, adherent, and nonadherent cell populations from spleen, peritoneal cavity, and mesenteric lymph node. The natural killer (NK) cell activity was measured in a 4-hr 51Cr-release assay with a xenogeneic murine YAC lymphoma target cell line. In both and +/rnu rats the peritoneal cavity had the highest percentage of activity, while the spleen and mesenteric lymph node showed a lower activity. The mesenteric lymph node of +/rnu rats of 8–10 weeks of age was found to express a very low activity, in contrast to a very high activity in rats. For almost every effector to target cell (E:T) ratio investigated (100, 70, 50, and 10), the natural killer cell activity in the nude rats was found to be significantly higher than in their thymus-bearing littermates. In comparison with that of +/rnu rats, NK activity in the nonadherent cell fractions of athymic rats was 50 to 60% higher in spleen cells, doubled in peritoneal cells, and increased 10-fold or higher in lymph node cells. Investigations o