Regulation of the expression of {\it mutS, mutL}, and {\it mutH\/} mismatch repair genes in {\it Escherichia coli\/} K-12

Post-replication DNA mismatch repair plays crucial roles in mutation avoidance and maintenance of chromosome stability in both prokaryotes and eukaryotes. In humans, deficiency in this repair system leads to a predisposition for certain cancers. The biochemistry of this repair system has been best studied in a model bacterium Escherichia coli. In this thesis, regulation of expression of mutS, mutL and mutH genes, whose products mediate methyl-directed mismatch (MDM) repair in E. coli, is investigated. One-step affinity purification schemes were developed to purify E. coli MutS, MutL and MutH proteins fused to a His-6-affinity tag. His-6-MutS exhibited the same mismatch binding activity and specificity as the native MutS protein. Purified His-6-MutS, -MutL and -MutH proteins were used to develop quantitative Western blotting assays for amounts of MutS, MuL and MutH proteins under various conditions. It was found that the three proteins were present in relatively low amounts in exponentially growing cells and MutS and MutH were diminished in stationary-phase cells. Further studies indicated that the drop in the amounts of MutS and MutH proteins in stationary-phase cells was mediated through RpoS, a key global regulator of stationary-phase transition. In both exponential- and stationary-phase cells, MutS amount was also negatively regulated by the Hfq (HF-I) global regulator, which is required for RpoS translation, through an RpoS-independent mechanism. $\beta$-galactosidase assays of mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally, and RNase T2 protection assays revealed that Hfq destabilizes mutS transcripts in exponentially growing cells. To study the relation between regulation of MDM repair and mutagenesis, amounts of MutS, MutL and MutH were measured in starved cells undergoing adaptive mutagenesis. It was found that MutS amount dropped drastically, MutH amount dropped slightly, whereas MutL amount remained essentially constant in starved cells. Overexpression of MutL did not reverse the drop in the amounts of MutS or MutH protein. These results ruled out several explanations for a phenomenon in which overexpression of MutL, but not MutS, reversed adaptive mutagenesis. The findings further suggested that functional MutL is limiting during adaptive mutagenesis. The implications of regulation of the MDM repair are discussed in the context of mutagenesis, pathogenesis and tumorigenesis. ^

Long-lasting synaptic potentiation induced by depolarization under conditions that eliminate detectable Ca2+ signals.

Activity-dependent alterations of synaptic transmission important for learning and memory are often induced by Ca(2+) signals generated by depolarization. While it is widely assumed that Ca(2+) is the essential transducer of depolarization into cellular plasticity, little effort has been made to test whether Ca(2+)-independent responses to depolarization might also induce memory-like alterations. It was recently discovered that peripheral axons of nociceptive sensory neurons in Aplysia display long-lasting hyperexcitability triggered by conditioning depolarization in the absence of Ca(2+) entry (using nominally Ca(2+)-free solutions containing EGTA, "0Ca/EGTA") or the absence of detectable Ca(2+) transients (adding BAPTA-AM, "0Ca/EGTA/BAPTA-AM"). The current study reports that depolarization of central ganglia to approximately 0 mV for 2 min in these same solutions induced hyperexcitability lasting >1 h in sensory neuron processes near their synapses onto motor neurons. Furthermore, conditioning depolarization in these solutions produced a 2.5-fold increase in excitatory postsynaptic potential (EPSP) amplitude 1-3 h afterward despite a drop in motor neuron input resistance. Depolarization in 0 Ca/EGTA produced long-term potentiation (LTP) of the EPSP lasting > or = 1 days without changing postsynaptic input resistance. When re-exposed to extracellular Ca(2+) during synaptic tests, prior exposure to 0Ca/EGTA or to 0Ca/EGTA/BAPTA-AM decreased sensory neuron survival. However, differential effects on neuronal health are unlikely to explain the observed potentiation because conditioning depolarization in these solutions did not alter survival rates. These findings suggest that unrecognized Ca(2+)-independent signals can transduce depolarization into long-lasting synaptic potentiation, perhaps contributing to persistent synaptic alterations following large, sustained depolarizations that occur during learning, neural injury, or seizures.

Three-dimensional electron beam dose calculations

The MDAH pencil-beam algorithm developed by Hogstrom et al (1981) has been widely used in clinics for electron beam dose calculations for radiotherapy treatment planning. The primary objective of this research was to address several deficiencies of that algorithm and to develop an enhanced version. Two enhancements have been incorporated into the pencil-beam algorithm; one models fluence rather than planar fluence, and the other models the bremsstrahlung dose using measured beam data. Comparisons of the resulting calculated dose distributions with measured dose distributions for several test phantoms have been made. From these results it is concluded (1) that the fluence-based algorithm is more accurate to use for the dose calculation in an inhomogeneous slab phantom, and (2) the fluence-based calculation provides only a limited improvement to the accuracy the calculated dose in the region just downstream of the lateral edge of an inhomogeneity. The source of the latter inaccuracy is believed primarily due to assumptions made in the pencil beam's modeling of the complex phantom or patient geometry.^ A pencil-beam redefinition model was developed for the calculation of electron beam dose distributions in three dimensions. The primary aim of this redefinition model was to solve the dosimetry problem presented by deep inhomogeneities, which was the major deficiency of the enhanced version of the MDAH pencil-beam algorithm. The pencil-beam redefinition model is based on the theory of electron transport by redefining the pencil beams at each layer of the medium. The unique approach of this model is that all the physical parameters of a given pencil beam are characterized for multiple energy bins. Comparisons of the calculated dose distributions with measured dose distributions for a homogeneous water phantom and for phantoms with deep inhomogeneities have been made. From these results it is concluded that the redefinition algorithm is superior to the conventional, fluence-based, pencil-beam algorithm, especially in predicting the dose distribution downstream of a local inhomogeneity. The accuracy of this algorithm appears sufficient for clinical use, and the algorithm is structured for future expansion of the physical model if required for site specific treatment planning problems. ^

Evaluation of the LBJ Hospital Ask Your Nurse Advice Line

This retrospective, case-control study investigated the effectiveness of the Houston, Texas Ask Your Nurse Advice Line (AYN) in diverting callers with non-emergent medical conditions away from the emergency department (ED). After asking callers a series of questions, AYN nurses evaluate the medical condition and make recommendations for appropriate care, e.g., home care, see a clinic physician, or visit the ED. To evaluate the AYN, the rate of caller ED visits before accessing the AYN for the first time was compared to the caller ED visit rate afterwards. The pre-post rate change was compared to that of a control group of similar caller age, race, gender, and insurance status drawn from a Harris County Hospital District HCHD database. ^ The treatment group (AYN caller) had a 66% reduction in ED visits after the first AYN call compared to an 18% drop in ED visits among control group subjects during the same time period. Study results were presented to HCHD staff on August 30th, 2007 and recommendations were made for future studies that would provide a basis for policy development. ^

Screening for anemia in Red Cross blood donor clinics

Accurate screening for anemia at Red Cross blood donor clinics is essential to maintain a safe national blood supply. Despite the importance of identifying anemia correctly by measurement of hemoglobin or hematocrit (hemoglobin/hematocrit) there is no consensus regarding the efficacy of the current two stage screening method which uses the Readacrit$\sp{\rm tm}$ microhematocrit in conjunction with copper sulfate.^ A cross-sectional study was implemented in which hemoglobin/hematocrit was measured, with the present method and four new devices, on 504 prospective blood donors at a Canadian Red Cross permanent blood donor clinic in London, Canada. Concurrently gathered, venous and capillary blood samples were tested by each device and compared to Coulter S IV$\sp{\rm tm}$ determined venous standard readings. Instrument hemoglobin/hematocrit means were statistically calibrated to the standard ones in order to appraise systematic deviations from the standard. Classification analysis was employed to assess concordance between each instrument and the standard when classifying prospective donors as anemic or non-anemic. This was done both when each instrument was used alone (single stage) and when copper sulfate was used as a preliminary screen (two stage) and simulated over a range of anemia prevalences. The Hemoximeter$\sp{\rm tm}$ and Compur M1000$\sp{\rm tm}$ devices had the highest correlations of hemoglobin measurements with the standard ones for both capillary (n.s.) and venous blood (p $<$.05). Analysis of variance (anova) also showed them to be the most accurate (p $<$.05), as did both single and two stage classification analysis, therefore, they are both recommended. There was a smaller difference between instruments for two stage than for single stage screening; therefore instrument choice is less crucial for the former. The present method was adequate for two stage screening as tested but simulations showed that it would discriminate poorly in populations with a higher prevalence of anemia. The Stat-crit and Readacrit, which measure hematocrit, became less accurate at crucial low hematocrit levels. In light of this finding and the introduction of new, effective and easy to use hemoglobin measuring instruments, the continued use of hematocrit as a surrogate for hemoglobin, is not recommended. ^

Costs and quality of medication reconciliation practice in primary care clinics

Medication reconciliation, with the aim to resolve medication discrepancy, is one of the Joint Commission patient safety goals. Medication errors and adverse drug events that could result from medication discrepancy affect a large population. At least 1.5 million adverse drug events and $3.5 billion of financial burden yearly associated with medication errors could be prevented by interventions such as medication reconciliation. This research was conducted to answer the following research questions: (1a) What are the frequency range and type of measures used to report outpatient medication discrepancy? (1b) Which effective and efficient strategies for medication reconciliation in the outpatient setting have been reported? (2) What are the costs associated with medication reconciliation practice in primary care clinics? (3) What is the quality of medication reconciliation practice in primary care clinics? (4) Is medication reconciliation practice in primary care clinics cost-effective from the clinic perspective? Study designs used to answer these questions included a systematic review, cost analysis, quality assessments, and cost-effectiveness analysis. Data sources were published articles in the medical literature and data from a prospective workflow study, which included 150 patients and 1,238 medications. The systematic review confirmed that the prevalence of medication discrepancy was high in ambulatory care and higher in primary care settings. Effective strategies for medication reconciliation included the use of pharmacists, letters, a standardized practice approach, and partnership between providers and patients. Our cost analysis showed that costs associated with medication reconciliation practice were not substantially different between primary care clinics using or not using electronic medical records (EMR) ($0.95 per patient per medication in EMR clinics vs. $0.96 per patient per medication in non-EMR clinics, p=0.78). Even though medication reconciliation was frequently practiced (97-98%), the quality of such practice was poor (0-33% of process completeness measured by concordance of medication numbers and 29-33% of accuracy measured by concordance of medication names) and negatively (though not significantly) associated with medication regimen complexity. The incremental cost-effectiveness ratios for concordance of medication number per patient per medication and concordance of medication names per patient per medication were both 0.08, favoring EMR. Future studies including potential cost-savings from medication features of the EMR and potential benefits to minimize severity of harm to patients from medication discrepancy are warranted. ^