Tego - A framework for adversarial planning

This study establishes a framework called ∗-Tego for a situation in which two agents are each given a set of players for a competitive game. Each agent places their players in an order. Players on each side at the same position in the order play one another, with the agent\u27s score being the sum of their player\u27s scores. The planning agents are permitted to simultaneous reorder their players in each of several stages. The reordering is termed competitive replanning. The resulting framework is scalable by changing the number of players and the complexity of the replanning process. The framework is demonstrated using iterated prisoner\u27s dilemma on a set of twenty players. The system is first tested with one agent unable to change the order of its players, yielding an optimization problem. The system is then tested in a competitive co-evolution of planning agents. The optimization form of the system makes globally sensible assignments of players. The co-evolutionary version concentrates on matching particular high-payoff pairs of players with the agents repeatedly reversing one another\u27s assignments, with the majority of players with smaller payoffs at risk are largely ignored

In vivo Electroporation and Non-protein Based Screening Assays to Identify Antibodies Against Native Protein Conformations

In vivo electroporation has become a gold standard method for DNA immunization. The method assists the DNA entry into cells, results in expression and the display of the native form of antigens to professional cells of the immune system, uses both arms of immune system, has a built-in adjuvant system, is relatively safe, and is cost-effective. However, there are challenges for achieving an optimized reproducible process for eliciting strong humoral responses and for the screening of specific immune responses, in particular, when the aim is to mount humoral responses or to generate monoclonal antibodies via hybridoma technology. Production of monoclonal antibodies demands generation of high numbers of primed B and CD4 T helper cells in lymphoid organs needed for the fusion that traditionally is achieved by a final intravenous antigen injection. The purified antigen is also needed for screening of hundreds of clones obtained upon fusion of splenocytes. Such challenges make DNA vaccination dependent on purified proteins. Here, we have optimized methods for in vivo electroporation, production, and use of cells expressing the antigen and an in-cell Western screening method. These methods resulted in (1) reproducibly mounting robust humoral responses against antigens with different cell localizations, and (2) the ability to screen for antigen eliminating a need for protein/antigen purification. This process includes optimized parameters for in vivo electroporation, the use of transfected cells for final boost, and mild fixation/permeabilization of cells for screening. Using this process, upon two vaccinations via in vivo electroporation (and final boost), monoclonal antibodies against nucleus and cytoplasmic and transmembrane proteins were achieved

In vivo

In vivo electroporation has become a gold standard method for DNA immunization. The method assists the DNA entry into cells, results in expression and the display of the native form of antigens to professional cells of the immune system, uses both arms of immune system, has a built-in adjuvant system, is relatively safe, and is cost-effective. However, there are challenges for achieving an optimized reproducible process for eliciting strong humoral responses and for the screening of specific immune responses, in particular, when the aim is to mount humoral responses or to generate monoclonal antibodies via hybridoma technology. Production of monoclonal antibodies demands generation of high numbers of primed B and CD4 T helper cells in lymphoid organs needed for the fusion that traditionally is achieved by a final intravenous antigen injection. The purified antigen is also needed for screening of hundreds of clones obtained upon fusion of splenocytes. Such challenges make DNA vaccination dependent on purified proteins. Here, we have optimized methods for in vivo electroporation, production, and use of cells expressing the antigen and an in-cell Western screening method. These methods resulted in (1) reproducibly mounting robust humoral responses against antigens with different cell localizations, and (2) the ability to screen for antigen eliminating a need for protein/antigen purification. This process includes optimized parameters for in vivo electroporation, the use of transfected cells for final boost, and mild fixation/permeabilization of cells for screening. Using this process, upon two vaccinations via in vivo electroporation (and final boost), monoclonal antibodies against nucleus and cytoplasmic and transmembrane proteins were achieved

Multicenter Intestinal Current Measurements in Rectal Biopsies from CF and Non-CF Subjects to Monitor CFTR Function

<div><p>Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations). All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy) or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (µA/cm²; 95% confidence intervals) between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1), carbachol=96.3(118.7 to 73.9), forskolin/IBMX+carbachol=200.9(243.1 to 158.6), and bumetanide=-44.6 (-33.7 to -55.6) (<i>P</i><0.005, CF vs non-CF for all parameters). Receiver Operating Characteristic curves indicated that each parameter discriminated CF from non-CF subjects (area under the curve of 0.94-0.98). CFTR dependent currents following 18-24 hours of cold storage for forskolin/IBMX, carbachol, and forskolin/IBMX+carbachol stimulation (n=17 non-CF subjects) were 44%, 47.5%, and 47.3%, respectively of those in fresh biopsies. CFTR-dependent currents from biopsies studied after cold storage at two sites simultaneously demonstrated moderate correlation (n=14 non-CF subjects, Pearson correlation coefficients 0.389, 0.484, and 0.533). Similar CFTR dependent currents were detected from fresh biopsies obtained by either forceps or suction (within-subject comparisons, n=22 biopsies from three non-CF subjects). Multicenter ICM is a feasible CFTR outcome measure that discriminates CF from non-CF ion transport, offers unique advantages over other CFTR bioassays, and warrants further development as a potential CFTR biomarker.</p> </div

ROC curve analysis.

<p>Note that CF participants are coded as one, and non-CF participants are coded as zero. Average true-positive rate is the sensitivity of the current to detect CF participants, whereas the average-false positive rate (one-specificity) marks the cutoff whereby non-CF participants are falsely determined as CF. AUC values varied from 0.946-0.978 for the three CFTR-specific measurements (forskolin/IBMX (cAMP), carbachol (CCh), cAMP + CCh).</p

Partial CFTR function detected in F508del/F508del CF subject.

<p>Representative tracing from an F508del/F508del subject with functional F508del CFTR. Adding forskolin/IBMX to raise cAMP increased the current > 30µA/cm² which was further augmented by carbachol.</p

Bland-Altman plots of ICM responses for biopsies from single subjects studied at two sites simultaneously after cold storage.

<p>Difference between <b>A</b>. forskolin/IBMX (cAMP), <b>B</b>. carbachol, or <b>C</b>. forskolin/IBMX (cAMP + carbachol) responses at site of biopsy origin and test site. Each dot is the mean ICM response for one subject (both sites). The X axis is the mean of the response (both sites), and the Y axis is the difference between the means at the two sites. The red line is the hypothetical zero difference, and the green line is the actual mean difference. Blue lines are ± 2 SDs.</p

Individual ICM responses across study sites.

<p>Boxplots of CFTR responses in non-CF subjects segregated by site (site identifiers S032, S076, S191). Whiskers are minimum and maximum values, boxes included data within 25^th-75^th percentiles, the horizontal line is the median, and the diamond is the mean. <b>A</b>. Change in I_sc following cAMP stimulation (10 µM forskolin/100 µM IBMX). <b>B</b>. Change in I_sc following carbachol (CCh) stimulation (100 µM, basolateral). <b>C</b>. Change in I_sc following cAMP + CCh stimulation (10 µM forskolin, 100 µM IBMX, 100 µM CCh).</p