Quantitation of C4a and C4b in systemic lupus erythematosus patients

The fourth component of human complement (C4) exists in blood as two major forms or isotypes which differ in their biochemical and functional properties. Because C4A preferentially transacylates onto amino groups, it has been postulated that this isotype is more important in the clearance of immune complexes. Patients having systemic lupus erythematosus (SLE), an autoimmune disease, have an increased incidence of C4A null genes and presumably decreased levels of C4A. Currently accepted methods for the detection of C4, however, cannot accurately quantitate C4A and C4B. Thus, their role in disease susceptibility and activity has not been studied. A novel immunoassay, which utilized heat-aggregated IgG to activate and capture C4, was developed for accurate quantitation of total C4, C4A and C4B by monoclonal antibody conjugates. Higher mean total C4 values were found in a healthy Black control population when compared to White controls. This appeared to be due to an increase in C4B. In SLE patients, mean total C4 levels were significantly lower than controls regardless of disease activity. Serial patient studies showed that the ratio of C4A:C4B remained relatively constant. When the patient group was compared to controls based on C4 null gene status, the mean levels of C4A were identical while C4B was decreased in the patients. This suggests that the common HLA-B8, Dr3 C4A*Q0 gene deletion found in SLE patients may also adversely affect genetic control of the C4B genes. Furthermore, low levels of C4A cannot fully account for disease development in SLE patients having C4A null genes. ^

Blended Learning: Integrating an e-Learning Component for Competency Validation

Introduction: Emergency care providers are required to demonstrate competency in the management of life-threatening situation. The care provider’s ability to manage an emergency situation depends upon his/her knowledge and skills in basic CPR; and the use of emergency equipment and supplies. The education department at our healthcare facility is responsible for CPR/Emergency Management competency validation of over 2500 employees annually. Historically each employee was scheduled to attend 4 hours of class every year to review the content, complete the post-test and demonstrate skills. It was resource-intensive, time consuming, stressful and often difficult to schedule the 24/7 employees for the sessions. [See PDF for complete abstract]

Cardiomyocyte PDGFR-beta signaling is an essential component of the mouse cardiac response to load-induced stress.

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

The characterization of an epithelial chloride channel and purification of a channel component

A membrane fraction (M$\sb{\rm PS}$), enriched in Cl$\sp-$ channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The tracheal fraction shows a 37 fold enrichment of Cl$\sp-$ channels over crude tracheal homogenates by net Cl$\sp-$ measurements in membrane vesicles. Alkaline phosphatase and (Na$\sp+$ + K$\sp+$)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. The M$\sb{\rm PS}$ fraction exhibits a protein profile unlike that of other membrane fractions with major proteins of 200 kDa and 42 kDa, proteins of 30 to 35 kDa, and lesser amounts of other proteins. Reconstitution of M$\sb{\rm PS}$ fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is linear with a slope conductance of 84 pS in symmetrical 400 mM KCl, and is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (Valdivia, Dubinsky, and Coronado. Science, 1988). In addition, the voltage dependence, selectivity sequence of Cl$\sp- >$ Br$\sp- \ge$ I$\sp-$, and inhibition by low concentrations of the Cl$\sp-$ channel blocker, DIDS, correspond to those of the predominant apical membrane channel. Thus, although the M$\sb{\rm PS}$ fraction appears to be of subcellular origin, it may be functionally related to an apical membrane Cl$\sp-$ permeability. When renal M$\sb{\rm PS}$ membranes were treated with the detergent octyl-glucoside (OG, 2%) and centrifuged, the supernatant, sM$\sb{\rm PS}$, showed a 2 to 7-fold enrichment in specific Cl$\sp-$ flux activity compared with the detergent treated M$\sb{\rm PS}$. These solubilized proteins were then size fractionated on a Superose 12 HPLC gel filtration column, followed by fractionation on a Mono Q HPLC anion exchange column. Fractions that eluted in high salt consistently exhibited significant Cl$\sp-$ flux activity. These fractions had protein profiles consisting of a major band at 34 kDa, a band at 66 kDa, and variable faint bands. Fractions eluting in lower salt had protein profiles consisting of a single band at 34 kDa, and often had little or no Cl$\sp-$ flux activity. However, co-reconstitution of the low salt, solely 34 kDa protein-containing Mono Q fractions with sM$\sb{\rm PS}$ resulted in an enhancement of flux activity compared to that of sM$\sb{\rm PS}$ reconstituted alone. Flux assays of active Mono Q fractions showed that the channel retained its DIDS sensitivity. Applying sM$\sb{\rm PS}$ to a DIDS-affinity column and eluting with salt resulted in fractions with protein profiles again consisting of at least one major band at 34 kDa, a band at 66 kDa, and variable faint bands. Co-reconstitution with sM$\sb{\rm PS}$ again resulted in an enhancement of activity. Thus, the 34 kDa protein appears to be a component of the M$\sb{\rm PS}$ Cl$\sp-$ channel. ^

Design and evaluation of the parent education component of a comprehensive violence prevention project in middle schools

Low parental monitoring is related to youth risk behaviors such as delinquency and aggression. The purpose of this dissertation was to describe the development and evaluation of a parent education intervention to increase parental monitoring in Hispanic parents of middle school children.^ The first study described the process of intervention mapping as used to develop Padres Trabajando por la Paz, a newsletter intervention for parents. Using theory, empirical literature, and information from the target population, performance objectives and determinants for monitoring were defined. Learning objectives were specified and a staged social-cognitive approach was used to develop methods and strategies delivered through newsletters.^ The second study examined the outcomes of a randomized trial of the newsletter intervention. Outcome measures consisted of a general measure of monitoring, parent and child reports of monitoring behaviors targeted by the intervention, and psychosocial determinants of monitoring (self-efficacy, norms, outcome expectancies, knowledge, and beliefs). Seventy-seven parents completed the randomized trial, half of which received four newsletters over an eight-week period. Results revealed a significant interaction effect for baseline and treatment for parent's reports of norms for monitoring (p =.009). Parents in the experimental condition who scored low at baseline reported increased norms for monitoring at follow-up. A significant interaction effect for child reports of parental monitoring behaviors (p =.04) reflected an small increase across baseline levels in the experimental condition and decreases for the control condition at higher baseline scores. Both groups of parents reported increased levels of monitoring at follow-up. No other outcome measures varied significantly by condition.^ The third study examined the relationship between the psychosocial determinants of parental monitoring and parental monitoring behaviors in the study population. Weak evidence for a relationship between outcome expectancies and parental monitoring behaviors suggests further research in the area utilizing stronger empirical models such as longitudinal design and structural equation modeling.^ The low-cost, minimal newsletter intervention showed promise for changing norms among Hispanic parents for parental monitoring. In light of the importance of parental monitoring as a protective factor for youth health risk behaviors, more research needs to be done to develop and evaluate interventions to increase parental monitoring. ^

Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

The School Sealant Program: An evaluation of the oral health education component

Dental caries is a common preventable childhood disease leading to severe physical, mental and economic repercussions for children and their families if left untreated. A needs assessment in Harris County reported that 45.9% of second graders had untreated dental caries. In order to address this growing problem, the School Sealant Program (SSP), a primary preventive initiative, was launched by the Houston Department of Health and Human Services (HDHHS) to provide oral health education, and underutilized dental preventive services to second grade children from participating Local School Districts (LSDs). ^ To determine the effectiveness and efficiency of the SSP, a program evaluation was conducted by the HDHHS between September 2007 and June 2008 for the Oral Health Education (OHE) component of the SSP. The objective of the evaluation was to assess short term changes in oral health knowledge of the participants and determine if these changes, if any, were due to the OHE sessions. An 8-item multiple choice pre/post test was developed for this purpose and administered to the participants before and immediately after the OHE sessions. ^ The present project analyzed pre and post test data of 1,088 second graders from 22 participating schools. Changes in overall and topic-specific knowledge of the program participants before and after the OHE sessions were analyzed using the Wilcoxon's signed rank test. ^ Results. The overall knowledge assessment showed a statistically significant (p <0.001) increase in the dental health knowledge of the participants after the oral health education sessions. Participants in the higher scoring category (7-8 correct responses) increased from 9.5% at baseline to 60.8% after the education sessions. Overall knowledge increased in all school regions with the highest knowledge gains seen in the Central and South regions. Males and females had similar knowledge gains. Significant knowledge differences were also found for each of the topic specific categories (functions of teeth, healthy diet, healthy habits, dental sealants; p<0.001) indicating an increase in topic specific knowledge of the participants post-health education sessions. ^ Conclusions. The OHE sessions were successful in increasing the short term oral health knowledge of the participants. ^

MODULATION OF HOST INTESTINAL MYOELECTRIC ACTIVITY BY LOCAL ANAPHYLAXIS: A COMPONENT OF PROTECTIVE IMMUNITY TO AN ENTERIC PARASITE (TRICHINELLA SPIRALIS, EIMERIA NIESCHULZI, BOWEL MOTILITY)

The hypothesis tested was that rapid rejection of Trichinella spiralis infective larvae from immunized rats following a challenge infection is associated with a local anaphylactic reaction, and this response should be reflected in altered small intestinal motility. The objective was to determine if altered gut smooth muscle function accompanies worm rejection based on the assumption that anaphylaxis in vivo could be detected by changes in intestinal smooth muscle contractile activity (ie. an equivalent of the Schultz-Dale reaction or in vitro anaphylaxis). The aims were to (1) characterize motility changes by monitoring intestinal myoelectric activity in conscious rats during the enteric phase of T. spiralis infection in immunized hosts, (2) detect the onset and magnitude of myoelectric changes caused by challenge infection in immunized rats, (3) determine the parasite stimulus causing changes, and (4) determine the specificity of host response to stimulation. Electrical slow wave frequency, spiking activity, normal interdigestive migrating myoelectric complexes and abnormal migrating action potential complexes were measured. Changes in myoelectric parameters induced by larvae inoculated into the duodenum of immune hosts differed from those associated with primary infection with respect to time of onset, magnitude and duration. Myoelectric changes elicited by live larvae could not be reproduced by inoculation of hosts with dead larvae, larval excretory-secretory products, or by challenge with a heterologous parasite, Eimeria nieschulzi. These results indicate that (1) local anaphylaxis is a component of the initial response to T. spiralis in immune hosts, since the rapid onset of altered smooth muscle function parallels in time the expression of rapid rejection of infective larvae, and (2) an active mucosal penetration attempt by the worm is necessary to elicit this host response. These findings provide evidence that worm rejection is a consequence of, or sequel to, an immediate hypersensitivity reaction elicited when parasites attempt to invade the gut mucosa of immunized hosts. ^

Familial component of epilepsy in cleft lip and palate

It is well recognized that offspring of women with epilepsy who are taking anticonvulsant medications have an increased incidence of clefting abnormalities. This increase has been attributed to the teratogenic effects of anticonvulsant medications but an alternative explanation involving a genetic association of epilepsy and clefting has also been proposed. Five family studies attempting to resolve this controversy have been inconclusive either because of study design or analytic limitations. This family study was designed to determine whether epilepsy aggregates in families ascertained by an individual with a clefting disorder. The Mayo Clinic medical linkage registry was used to identify individuals with cleft lip with or without cleft palate and cleft palate in southeast Minnesota from 1935-1986. Only those cases who were 15 years or younger during this period were included in the study. The proband's parents and descendants of their parents, including the proband's sibs, children, grandchildren, niece/nephews, grandnieces/nephews, halfsibs and spouses were also identified and all of their medical records were reviewed for seizure disorders. The standardized morbidity ratios for epilepsy of 0.9 (95% CI 0.2-2.6) observed for first degree relatives (excluding parents) and 0.0 for second degree relatives were not increased. The SMRs ranged from 0.7-2.2 for the individual relative types (parents 1.5, sibs 0.7, children 2.2, probands 1.1, spouses 2.0) and were also not increased. These results do not support the suggestions of some that clefting and epilepsy aggregate together in families. ^

The application of principal component analysis on human development indicators: Temporal approach from 1999 to 2010

Background and Objective. Ever since the human development index was published in 1990 by the United Nations Development Programme (UNDP), many researchers started searching and corporative studying for more effective methods to measure the human development. Published in 1999, Lai’s “Temporal analysis of human development indicators: principal component approach” provided a valuable statistical way on human developmental analysis. This study presented in the thesis is the extension of Lai’s 1999 research. ^ Methods. I used the weighted principal component method on the human development indicators to measure and analyze the progress of human development in about 180 countries around the world from the year 1999 to 2010. The association of the main principal component obtained from the study and the human development index reported by the UNDP was estimated by the Spearman’s rank correlation coefficient. The main principal component was then further applied to quantify the temporal changes of the human development of selected countries by the proposed Z-test. ^ Results. The weighted means of all three human development indicators, health, knowledge, and standard of living, were increased from 1999 to 2010. The weighted standard deviation for GDP per capita was also increased across years indicated the rising inequality of standard of living among countries. The ranking of low development countries by the main principal component (MPC) is very similar to that by the human development index (HDI). Considerable discrepancy between MPC and HDI ranking was found among high development countries with high GDP per capita shifted to higher ranks. The Spearman’s rank correlation coefficient between the main principal component and the human development index were all around 0.99. All the above results were very close to outcomes in Lai’s 1999 report. The Z test result on temporal analysis of main principal components from 1999 to 2010 on Qatar was statistically significant, but not on other selected countries, such as Brazil, Russia, India, China, and U.S.A.^ Conclusion. To synthesize the multi-dimensional measurement of human development into a single index, the weighted principal component method provides a good model by using the statistical tool on a comprehensive ranking and measurement. Since the weighted main principle component index is more objective because of using population of nations as weight, more effective when the analysis is across time and space, and more flexible when the countries reported to the system has been changed year after year. Thus, in conclusion, the index generated by using weighted main principle component has some advantage over the human development index created in UNDP reports.^

The SasS -SasR two-component signal transduction system required for early Myxococcus xanthus multicellular *development

Initiation of Myxococcus xanthus multicellular development requires both nutrient limitation and high cell density. The extracellular signal, A signal, which consists of a set of amino acids at specific concentrations, serves as a cell density signal in M. xanthus early development. A reporter gene, designated 4521, that requires both starvation and A signal for developmental expression was used to identify mutations in the signal transduction pathways. A group of point mutations located in the chromosomal sasB locus that bypasses both requirements was previously isolated. One of these point mutations, sasB7, was mapped to the sasS gene, which is predicted to encode a transmembrane histidine protein kinase required for normal development. SasS is a positive regulator of 4521 and a candidate A signal sensor. This dissertation continues the characterization of the sasB locus, focusing on the sasR gene and the functional relationship of SasS and SasR. ^ The sasR gene is located 2.2-kb downstream of sasS. It is predicted to encode an NtrC-like response regulator, which belongs to the family of sigma54 transcriptional activators. SasR is a positive regulator of 4521 gene and is required for normal development. The sasR mutant displays phenotypes similar to that of sasS mutant. Both SasS and SasR are required for the A-signal-dependent 4521 expression. Genetic epistasis analysis indicates that SasR functions downstream of SasS. Biochemical studies show that SasS has autokinase activity, and phosphorylated SasS is able to transfer its phosphate to SasR. We propose that SasS and SasR form a two-component signal transduction system in the A signal transduction pathway. ^ To search for the genes regulated by SasS and SasR, expression patterns of a group of developmental genes were compared in wild-type and sasS null mutant backgrounds. SasS and SasR were found to positively regulate sasN and 4521. The sasN gene was previously identified as a negative regulator of 4521, located at about 170-bp downstream of sasR. It is required for normal fruiting body development. Based on the above data, a regulatory network consisting of sasS, sasR, sasN, and 4521 is hypothesized, and the interactions of the components in this network can now be further studied. ^

Human complement component C3 -binding proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that uses the host mononuclear phagocyte as a niche for survival and replication during infection. Complement component C3 has previously been shown to enhance the binding of M. tuberculosis to mononuclear phagocytes. Using a C3 ligand affinity blot protocol, we identified a 30 kDa C3-binding protein in M. tuberculosis as heparin-binding hemagglutinin (HbhA). HbhA was found to be a hydrophobic protein that localized to the cell membrane/cell wall fraction of M. tuberculosis, and this protein has previously been shown by others to be located on the surface of M. tuberculosis. The C3-binding activity of HbhA was localized to the C-terminus of the protein, which consists of lysine-alanine repeats. Full-length recombinant HbhA coated onto latex beads was shown to mediate the adherence of the beads to murine macrophage-like cells in both a C3-dependent and a C3-independent manner. An in-frame 576 by deletion in the hbhA gene was created in a virulent strain of M. tuberculosis using a PCR technique known as gene splicing by overlap extension (SOEing). Using the ΔhbhA mutant, HbhA was found not to be necessary for growth of M. tuberculosis in laboratory media or in macrophage-like cells, nor is HbhA required for adherence of M. tuberculosis to macrophage-like cells. HbhA is, however, required for infectivity of M. tuberculosis in mice. Mice infected with the ΔhbhA mutant show decreased growth in the lungs, liver, and spleen compared to mice infected with the wild-type strain. Using the ΔhbhA mutant strain, we were able to purify and identify a second 30-kDa C3-binding protein, HupB. These data demonstrate that HbhA is required for the in vivo but not the in vitro survival of M. tuberculosis and that HbhA is not necessary for the adherence of M. tuberculosis to the macrophage-like cells used in these studies. The expression of two proteins that bind human C3 may aid in the efficient binding of M. tuberculosis to complement receptors for uptake into mononuclear cells, or may influence other aspects of the host-parasite interaction. ^