THEORETICAL STUDIES ON THE METHODS OF RECONSTRUCTING PHYLOGENETIC TREES FROM DNA SEQUENCE DATA (MOLECULAR EVOLUTION, HOMINOID EVOLUTION, COMPUTER SIMULATION)

(1) A mathematical theory for computing the probabilities of various nucleotide configurations is developed, and the probability of obtaining the correct phylogenetic tree (model tree) from sequence data is evaluated for six phylogenetic tree-making methods (UPGMA, distance Wagner method, transformed distance method, Fitch-Margoliash's method, maximum parsimony method, and compatibility method). The number of nucleotides (m*) necessary to obtain the correct tree with a probability of 95% is estimated with special reference to the human, chimpanzee, and gorilla divergence. m* is at least 4,200, but the availability of outgroup species greatly reduces m* for all methods except UPGMA. m* increases if transitions occur more frequently than transversions as in the case of mitochondrial DNA. (2) A new tree-making method called the neighbor-joining method is proposed. This method is applicable either for distance data or character state data. Computer simulation has shown that the neighbor-joining method is generally better than UPGMA, Farris' method, Li's method, and modified Farris method on recovering the true topology when distance data are used. A related method, the simultaneous partitioning method, is also discussed. (3) The maximum likelihood (ML) method for phylogeny reconstruction under the assumption of both constant and varying evolutionary rates is studied, and a new algorithm for obtaining the ML tree is presented. This method gives a tree similar to that obtained by UPGMA when constant evolutionary rate is assumed, whereas it gives a tree similar to that obtained by the maximum parsimony tree and the neighbor-joining method when varying evolutionary rate is assumed. ^

AN IMPLANTABLE MOSFET DOSIMETER MODIFIED TO ACT AS A FIDUCIAL MARKER

In external beam radiation therapy, it is imperative that the prescribed dose is administered to the correct location and in the correct amount. Though several ex vivo methods of quality assurance are currently employed to achieve this goal, verifying that the correct dose is received within the patient in situ is impossible without the capability of measuring dose inside the patient. Recently, a method of measuring dose delivered within the patient has been developed, an implantable MOSFET dosimeter. This dosimeter is implanted within the patient and records the dose received. Since the dosimeter is implanted in the patient, it could serve a dual function as a fiducial marker for image guided radiation therapy (IGRT) treatment if it could be modified to be visible on x-rays. In this study, modifications to the MOSFET dosimeter were made to increase its visibility for IGRT treatment. To test whether the modifications hindered the dosimeter’s ability to accurately measure and transmit dose information, the energy dependence, angular dependence and wireless read range of the modified dosimeter were measured and compared to unmodified dosimeters. It was found that the modified dosimeter performed as well as the unmodified dosimeter while also being suitable for use as a fiducial marker for IGRT treatment.

Fluorophotometry is a sensitive method/technique to evaluate epithelial permeability in murine experimental dry eye

Purpose. Fluorophotometry is a well validated method for assessing corneal permeability in human subjects. However, with the growing importance of basic science animal research in ophthalmology, fluorophotometry’s use in animals must be further evaluated. The purpose of this study was to evaluate corneal epithelial permeability following desiccating stress using the modified Fluorotron Master™. ^ Methods. Corneal permeability was evaluated prior to and after subjecting 6-8 week old C57BL/6 mice to experimental dry eye (EDE) for 2 and 5 days (n=9/time point). Untreated mice served as controls. Ten microliters of 0.001% sodium fluorescein (NaF) were instilled topically into each mouse’s left eye to create an eye bath, and left to permeate for 3 minutes. The eye bath was followed by a generous wash with Buffered Saline Solution (BSS) and alignment with the Fluorotron Master™. Seven corneal scans using the Fluorotron Master were performed during 15 minutes (1 st post-wash scans), followed by a second wash using BSS and another set of five corneal scans (2nd post-wash scans) during the next 15 minutes. Corneal permeability was calculated using data calculated with the FM™ Mouse software. ^ Results. When comparing the difference between the Post wash #1 scans within the group and the Post wash #2 scans within the group using a repeated measurement design, there was a statistical difference in the corneal fluorescein permeability of the Post-wash #1 scans after 5 days (1160.21±108.26 vs. 1000.47±75.56 ng/mL, P<0.016 for UT-5 day comparison 8 [0.008]), but not after only 2 days of EDE compared to Untreated mice (1115.64±118.94 vs. 1000.47±75.56 ng/mL, P>0.016 for UT-2 day comparison [0.050]). There was no statistical difference between the 2 day and 5 day Post wash #1 scans (P=.299). The Post-wash #2 scans demonstrated that EDE caused a significant NaF retention at both 2 and 5 days of EDE compared to baseline, untreated controls (1017.92±116.25, 1015.40±120.68 vs. 528.22±127.85 ng/mL, P<0.05 [0.0001 for both]). There was no statistical difference between the 2 day and 5 day Post wash #2 scans (P=.503). The comparison between the Untreated post wash #1 with untreated post wash #2 scans using a Paired T-test showed a significant difference between the two sets of scans (P=0.000). There is also a significant difference between the 2 day comparison and the 5 day comparison (P values = 0.010 and 0.002, respectively). ^ Conclusion. Desiccating stress increases permeability of the corneal epithelium to NaF, and increases NaF retention in the corneal stroma. The Fluorotron Master is a useful and sensitive tool to evaluate corneal permeability in murine dry eye, and will be a useful tool to evaluate the effectiveness of dry eye treatments in animal-model drug trials.^

A simulation study of the standard design, the rolling six design, the CRM, and the modified CRM in Phase I clinical trials

The Phase I clinical trial is considered the "first in human" study in medical research to examine the toxicity of a new agent. It determines the maximum tolerable dose (MTD) of a new agent, i.e., the highest dose in which toxicity is still acceptable. Several phase I clinical trial designs have been proposed in the past 30 years. The well known standard method, so called the 3+3 design, is widely accepted by clinicians since it is the easiest to implement and it does not need a statistical calculation. Continual reassessment method (CRM), a design uses Bayesian method, has been rising in popularity in the last two decades. Several variants of the CRM design have also been suggested in numerous statistical literatures. Rolling six is a new method introduced in pediatric oncology in 2008, which claims to shorten the trial duration as compared to the 3+3 design. The goal of the present research was to simulate clinical trials and compare these phase I clinical trial designs. Patient population was created by discrete event simulation (DES) method. The characteristics of the patients were generated by several distributions with the parameters derived from a historical phase I clinical trial data review. Patients were then selected and enrolled in clinical trials, each of which uses the 3+3 design, the rolling six, or the CRM design. Five scenarios of dose-toxicity relationship were used to compare the performance of the phase I clinical trial designs. One thousand trials were simulated per phase I clinical trial design per dose-toxicity scenario. The results showed the rolling six design was not superior to the 3+3 design in terms of trial duration. The time to trial completion was comparable between the rolling six and the 3+3 design. However, they both shorten the duration as compared to the two CRM designs. Both CRMs were superior to the 3+3 design and the rolling six in accuracy of MTD estimation. The 3+3 design and rolling six tended to assign more patients to undesired lower dose levels. The toxicities were slightly greater in the CRMs.^

Use of a modified delphi process for establishing occupational exposure limits in support of health surveillance for space travelers

On-orbit exposures can come from numerous factors related to the space environment as evidenced by almost 50 years of environmental samples collected for water analysis, air analysis, radiation analysis, and physiologic parameters. For astronauts and spaceflight participants the occupational exposures can be very different from those experienced by workers performing similar tasks in workplaces on Earth, because the duration of the exposure could be continuous for very long orbital, and eventually interplanetary, missions. The establishment of long-term exposure standards is vital to controlling the quality of the spacecraft environment over long periods. NASA often needs to update and revise its prior exposure standards (Spacecrafts Maximum Allowable Concentrations (SMACs)). Traditional standards-setting processes are often lengthy, so a more rapid method to review and establish standards would be a substantial advancement in this area. This project investigates use of the Delphi method for this purpose. ^ In order to achieve the objectives of this study a modified Delphi methodology was tested in three trials executed by doctoral students and a panel of experts in disciplines related to occupational safety and health. During each test/trial modifications were made to the methodology. Prior to submission of the Delphi Questionnaire to the panel of experts a pilot study/trial was conducted using five doctoral students with the goals of testing and adjusting the Delphi questionnaire to improve comprehension, work out any procedural issues and evaluate the effectiveness of the questionnaire in drawing the desired responses. The remainder of the study consisted of two trials of the Modified Delphi process using 6 chemicals that currently have the potential of causing occupational exposures to NASA astronauts or spaceflight participants. To assist in setting Occupational Exposure Limits (OEL), the expert panel was established consisting of experts from academia, government and industry. Evidence was collected and used to create close-ended questionnaires which were submitted to the Delphi panel of experts for the establishment of OEL values for three chemicals from the list of six originally selected (trial 1). Once the first Delphi trial was completed, adjustments were made to the Delphi questionnaires and the process above was repeated with the remaining 3 chemicals (trial 2). ^ Results indicate that experience in occupational safety and health and with OEL methodologies can have a positive effect in minimizing the time experts take in completing this process. Based on the results of the questionnaires and comparison of the results with the SMAC already established by NASA, we conclude that use of the Delphi methodology is appropriate for use in the decision-making process for the selection of OELs.^

Modified 3+3 design for phase I clinical trials

Phase I clinical trial is mainly designed to determine the maximum tolerated dose (MTD) of a new drug. Optimization of phase I trial design is crucial to minimize the number of enrolled patients exposed to unsafe dose levels and to provide reliable information to the later phases of clinical trials. Although it has been criticized about its inefficient MTD estimation, nowadays the traditional 3+3 method remains dominant in practice due to its simplicity and conservative estimation. There are many new designs that have been proven to generate more credible MTD estimation, such as the Continual Reassessment Method (CRM). Despite its accepted better performance, the CRM design is still not widely used in real trials. There are several factors that contribute to the difficulties of CRM adaption in practice. First, CRM is not widely accepted by the regulatory agencies such as FDA in terms of safety. It is considered to be less conservative and tend to expose more patients above the MTD level than the traditional design. Second, CRM is relatively complex and not intuitive for the clinicians to fully understand. Third, the CRM method take much more time and need statistical experts and computer programs throughout the trial. The current situation is that the clinicians still tend to follow the trial process that they are comfortable with. This situation is not likely to change in the near future. Based on this situation, we have the motivation to improve the accuracy of MTD selection while follow the procedure of the traditional design to maintain simplicity. We found that in 3+3 method, the dose transition and the MTD determination are relatively independent. Thus we proposed to separate the two stages. The dose transition rule remained the same as 3+3 method. After getting the toxicity information from the dose transition stage, we combined the isotonic transformation to ensure the monotonic increasing order before selecting the optimal MTD. To compare the operating characteristics of the proposed isotonic method and the other designs, we carried out 10,000 simulation trials under different dose setting scenarios to compare the design characteristics of the isotonic modified method with standard 3+3 method, CRM, biased coin design (BC) and k-in-a-row design (KIAW). The isotonic modified method improved MTD estimation of the standard 3+3 in 39 out of 40 scenarios. The improvement is much greater when the target is 0.3 other than 0.25. The modified design is also competitive when comparing with other selected methods. A CRM method performed better in general but was not as stable as the isotonic method throughout the different dose settings. The results demonstrated that our proposed isotonic modified method is not only easily conducted using the same procedure as 3+3 but also outperforms the conventional 3+3 design. It can also be applied to determine MTD for any given TTL. These features make the isotonic modified method of practical value in phase I clinical trials.^