Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.

Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.

Host risk factors for rifamycin-resistant clostridium difficile infection: A case control study

Objective. To evaluate the host risk factors associated with rifamycin-resistant Clostridium difficile (C. diff) infection in hospitalized patients compared to rifamycin-susceptible C.diff infection.^ Background. C. diff is the most common definable cause of nosocomial diarrhea affecting elderly hospitalized patients taking antibiotics for prolonged durations. The epidemiology of Clostridium difficile associated disease is now changing with the reports of a new hypervirulent strain causing hospital outbreaks. This new strain is associated with increased disease severity and mortality. The conventional therapy for C. diff includes metronidazole and vancomycin but high recurrence rates and treatment failures are now becoming a major concern. Rifamycin antibiotics are being developed as a new therapeutic option to treat C. diff infection after their efficacy was established in a few in vivo and in vitro studies. There are some recent studies that report an association between the hypervirulent strain and emerging rifamycin resistance. These findings assess the need for clinical studies to better understand the efficacy of rifamycin drugs against C. diff.^ Methods. This is a hospital-based, matched case-control study using de-identified data drawn from two prospective cohort studies involving C. diff patients at St Luke's Hospital. The C. diff isolates from these patients are screened for rifamycin resistance using agar dilution methods for minimum inhibitory concentrations (MIC) as part of Dr Zhi-Dong Jiang's study. Twenty-four rifamycin-rifamycin resistant C. diff cases were identified and matched with one rifamycin susceptible C. diff control on the basis of ± 10 years of age and hospitalization 30 days before or after the case. De-identified data for the 48 subjects was obtained from Dr Kevin Garey's clinical study at St Luke's Hospital enrolling C. diff patients. It was reviewed to gather information about host risk factors, outcome variables and relevant clinical characteristic.^ Results. Medical diagnosis at the time of admission (p = 0.0281) and history of chemotherapy (p = 0.022) were identified as a significant risk factor while hospital stay ranging from 1 week to 1 month and artificial feeding were identified as an important outcome variable (p = 0.072 and p = 0.081 respectively). Horn's Index assessing the severity of underlying illness and duration of antibiotics for cases and controls showed no significant difference.^ Conclusion. The study was a small project designed to identify host risk factors and understand the clinical implications of rifamycin-resistance. The study was underpowered and a larger sample size is needed to validate the results.^

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.

Tracking of plasma lipids and lipoproteins, within-person and year-to-year variability in plasma cholesterol levels among participants from age 8 to 18 in Project HeartBeat!

Coronary heart disease remains the leading cause of death in the United States and increased blood cholesterol level has been found to be a major risk factor with roots in childhood. Tracking of cholesterol, i.e., the tendency to maintain a particular cholesterol level relative to the rest of the population, and variability in blood lipid levels with increase in age have implications for cholesterol screening and assessment of lipid levels in children for possible prevention of further rise to prevent adulthood heart disease. In this study the pattern of change in plasma lipids, over time, and their tracking were investigated. Also, within-person variance and retest reliability defined as the square root of within-person variance for plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides and their relation to age, sex and body mass index among participants from age 8 to 18 years were investigated. ^ In Project HeartBeat!, 678 healthy children aged 8, 11 and 14 years at baseline were enrolled and examined at 4-monthly intervals for up to 4 years. We examined the relationship between repeated observations by Pearson's correlations. Age- and sex-specific quintiles were calculated and the probability of participants to remain in the uppermost quintile of their respective distribution was evaluated with life table methods. Plasma total cholesterol, HDL-C and LDL-C at baseline were strongly and significantly correlated with measurements at subsequent visits across the sex and age groups. Plasma triglyceride at baseline was also significantly correlated with subsequent measurements but less strongly than was the case for other plasma lipids. The probability to remain in the upper quintile was also high (60 to 70%) for plasma total cholesterol, HDL-C and LDL-C. ^ We used a mixed longitudinal, or synthetic cohort design with continuous observations from age 8 to 18 years to estimate within person variance of plasma total cholesterol, HDL-C, LDL-C and triglycerides. A total of 5809 measurements were available for both cholesterol and triglycerides. A multilevel linear model was used. Within-person variance among repeated measures over up to four years of follow-up was estimated for total cholesterol, HDL-C, LDL-C and triglycerides separately. The relationship of within-person and inter-individual variance with age, sex, and body mass index was evaluated. Likelihood ratio tests were conducted by calculating the deviation of −2log (likelihood) within the basic model and alternative models. The square root of within-person variance provided the retest reliability (within person standard deviation) for plasma total cholesterol, HDL-C, LDL-C and triglycerides. We found 13.6 percent retest reliability for plasma cholesterol, 6.1 percent for HDL-cholesterol, 11.9 percent for LDL-cholesterol and 32.4 percent for triglycerides. Retest reliability of plasma lipids was significantly related with age and body mass index. It increased with increase in body mass index and age. These findings have implications for screening guidelines, as participants in the uppermost quintile tended to maintain their status in each of the age groups during a four-year follow-up. The magnitude of within-person variability of plasma lipids influences the ability to classify children into risk categories recommended by the National Cholesterol Education Program. ^

Hepatitis B and C infections in U.S. incarcerated populations: Prevalence and related mortality

Published reports have consistently indicated high prevalence of serologic markers for hepatitis B (HBV) and hepatitis C (HCV) infection in U.S. incarcerated populations. Quantifying the current and projected burden of HBV and HCV infection and hepatitis-related sequelae in correctional healthcare systems with even modest precision remains elusive, however, because the prevalence and sequelae of HBV and HCV in U.S. incarcerated populations are not well-studied. This dissertation contributes to the assessment of the burden of HBV and HCV infections in U.S. incarcerated populations by addressing some of the deficiencies and gaps in previous research. ^ Objectives of the three dissertation studies were: (1) To investigate selected study-level factors as potential sources of heterogeneity in published HBV seroprevalence estimates in U.S. adult incarcerated populations (1975-2005), using meta-regression techniques; (2) To quantify the potential influence of suboptimal sensitivity of screening tests for antibodies to hepatitis C virus (anti-HCV) on previously reported anti-HCV prevalence estimates in U.S. incarcerated populations (1990-2005), by comparing these estimates to error-adjusted anti-HCV prevalence estimates in these populations; (3) To estimate death rates due to HBV, HCV, chronic liver disease (CLD/cirrhosis), and liver cancer from 1984 through 2003 in male prisoners in custody of the Texas Department of Criminal Justice (TDCJ) and to quantify the proportion of CLD/cirrhosis and liver cancer prisoner deaths attributable to HBV and/or HCV. ^ Results were as follows. Although meta-regression analyses were limited by the small body of literature, mean population age and serum collection year appeared to be sources of heterogeneity, respectively, in prevalence estimates of antibodies to HBV antigen (HBsAg+) and any positive HBV marker. Other population characteristics and study methods could not be ruled out as sources of heterogeneity. Anti-HCV prevalence is likely somewhat higher in male and female U.S. incarcerated populations than previously estimated in studies using anti-HCV screening tests alone without the benefit of repeat or additional testing. Death rates due to HBV, HCV, CLD/cirrhosis, and liver cancer from 1984 through 2003 in TDCJ male prisoners exceeded state and national rates. HCV rates appeared to be increasing and disproportionately affecting Hispanics. HCV was implicated in nearly one-third of liver cancer deaths. ^

Selection of body fat distribution indices in adults and associations with plasma lipid metabolism variables

Body fat distribution is a cardiovascular health risk factor in adults. Body fat distribution can be measured through various methods including anthropometry. It is not clear which anthropometric index is suitable for epidemiologic studies of fat distribution and cardiovascular disease. The purpose of the present study was to select a measure of body fat distribution from among a series of indices (those traditionally used in the literature and others constructed from the analysis) that is most highly correlated with lipid-related variables and is independent of overall fatness. Subjects were Mexican-American men and women (N = 1004) from a study of gallbladder disease in Starr County, Texas. Multivariate associations were sought between lipid profile measures (lipids, lipoproteins, and apolipoproteins) and two sets of anthropometric variables (4 circumferences and 6 skinfolds). This was done to assess the association between lipid-related measures and the two sets of anthropometric variables and guide the construction of indices.^ Two indices emerged from the analysis that seemed to be highly correlated with lipid profile measures independent of obesity. These indices are: 2*arm circumference-thigh skinfold in pre- and post-menopausal women and arm/thigh circumference ratio in men. Next, using the sum of all skinfolds to represent obesity and the selected body fat distribution indices, the following hypotheses were tested: (1) state of obesity and centrally/upper distributed body fat are equally predictive of lipids, lipoproteins and apolipoproteins, and (2) the correlation among the lipid-related measures is not altered by obesity and body fat distribution.^ With respect to the first hypothesis, the present study found that most lipids, lipoproteins and apolipoproteins were significantly associated with both overall fatness and anatomical location of body fat in both sex and menopausal groups. However, within men and post-menopausal women, certain lipid profile measures (triglyceride and HDLT among post-menopausal women and apos C-II, CIII, and E among men) had substantially higher correlation with body fat distribution as compared with overall fatness.^ With respect to the second hypothesis, both obesity and body fat distribution were found to alter the association among plasma lipid variables in men and women. There was a suggestion from the data that the pattern of correlations among men and post-menopausal women are more comparable. Among men correlations involving apo A-I, HDLT, and HDL$\sb2$ seemed greatly influenced by obesity, and A-II by fat distribution; among post-menopausal women correlations involving apos A-I and A-II were highly affected by the location of body fat.^ Thus, these data point out that not only can obesity and fat distribution affect levels of single measures, they also can markedly influence the pattern of relationship among measures. The fact that such changes are seen for both obesity and fat distribution is significant, since the indices employed were chosen because they were independent of one another. ^

Growth and development of body fat distribution from skinfold measurements: Longitudinal principal components

Longitudinal principal components analyses on a combination of four subcutaneous skinfolds (biceps, triceps, subscapular and suprailiac) were performed using data from the London Longitudinal Growth Study. The main objectives were to discover at what age during growth sex differences in body fat distribution occur and to see if there is continuity in body fatness and body fat distribution from childhood into the adult status (18 years). The analyses were done for four age sectors (3mon-3yrs, 3yrs-8yrs, 8yrs-18yrs and 3yrs-18yrs). Longitudinal principal component one (LPC1) for each age interval in both sexes represents the population mean fat curve. Component two (LPC2) is a velocity of fatness component. Component three (LPC3) in the 3mon-3yrs age sector represents infant fat wave in both sexes. In the next two age sectors component three in males represents peaks and shifts in fat growth (change in velocity), while in females it represents body fat distribution. Component four (LPC4) in the same two age sectors is a reversal in the sexes of the patterns seen for component three, i.e., in males it is body fat distribution and in females velocity shifts. Components five and above represent more complicated patterns of change (multiple increases and decreases across the age interval). In both sexes there is strong tracking in fatness from middle childhood to adolescence. In males only there is also a low to moderate tracking of infant fat with middle to late childhood fat. These data are strongly supported in the literature. Several factors are known to predict adult fatness among the most important being previous levels of fatness (at earlier ages) and the age at rebound. In addition we found that the velocity of fat change in middle childhood was highly predictive of later fatness (r $\approx -$0.7), even more so than age at rebound (r $\approx -$0.5). In contrast to fatness (LPC1), body fat distribution (LPC3-LPC4) did not track well even though significant components of body fat distribution occur at each age. Tracking of body fat distribution was higher in females than males. Sex differences in body fat distribution are non existent. Some sex differences are evident with the peripheral-to-central ratios after age 14 years. ^