PURINERGIC SIGNALING REGULATES FILOPODIA-INDUCED ZIPPERING

The molecular mechanisms that mediate endometrial cancer invasion and metastasis remain poorly understood. This is a significant clinical problem, as there is no definitive cure for metastatic disease. The purinergic pathway’s generation of adenosine and its activation of the adenosine receptor A2B (A2BR) induces cell-cell adhesion to promote barrier function. This barrier function is known to be important in maintaining homeostasis during hypoxia, trauma, and sepsis. Loss of this epithelial barrier function provides a considerable advantage for carcinoma progression, as loss of cell-cell adhesions supports proliferation, aberrant signaling, epithelial-to-mesenchymal transition, invasion, and metastasis. The present work provides strong evidence that CD73-generated adenosine actively promotes cell-cell adhesion in carcinoma cells by filopodia-induced zippering. Adenosine-generating ecto-enzyme, CD73, was down-regulated in moderately- and poorly-differentiated, invasive, and metastatic endometrial carcinomas. CD73 expression and enzyme activity in normal endometrium and endometrial carcinomas was significantly correlated to the epithelial phenotype. Barrier function in normal epithelial cells of the endometrium was dependent on stress-induced generation of adenosine by CD73 and adenosine’s activation of A2BR. This same mechanism inhibited endometrial carcinoma cell migration and invasion. Finally, adenosine’s activation of A2BR induced the formation of filopodia that promoted the re-forming of cell-cell adhesions in carcinoma cells. Overall, these studies identified purinergic pathway-induced filopodia to be a novel mechanism of adenosine’s barrier function and a mechanism that has to be avoided/down-regulated by endometrial carcinoma cells attempting to lose attachment with their neighboring cells. These results provide insight into the molecular mechanisms of endometrial cancer invasion. In addition, because loss of cell-cell adhesions has been closely linked to therapy resistance in cancer, these results provide a rational clinical strategy for the re-establishment of cell-cell adhesions to potentially increase therapeutic sensitivity. In contrast to other molecular mechanisms regulating cell-cell adhesions, the purinergic pathway is clinically druggable, with agonists and antagonists currently being tested in clinical trials of various diseases.

Survival analysis of longevity in siblings

Do siblings of centenarians tend to have longer life spans? To answer this question, life spans of 184 siblings for 42 centenarians have been evaluated. Two important questions have been addressed in analyzing the sibling data. First, a standard needs to be established, to which the life spans of 184 siblings are compared. In this report, an external reference population is constructed from the U.S. life tables. Its estimated mortality rates are treated as baseline hazards from which the relative mortality of the siblings are estimated. Second, the standard survival models which assume independent observations are invalid when correlation within family exists, underestimating the true variance. Methods that allow correlations are illustrated by three different methods. First, the cumulative relative excess mortality between siblings and their comparison group is calculated and used as an effective graphic tool, along with the Product Limit estimator of the survival function. The variance estimator of the cumulative relative excess mortality is adjusted for the potential within family correlation using Taylor linearization approach. Second, approaches that adjust for the inflated variance are examined. They are adjusted one-sample log-rank test using design effect originally proposed by Rao and Scott in the correlated binomial or Poisson distribution setting and the robust variance estimator derived from the log-likelihood function of a multiplicative model. Nether of these two approaches provide correlation estimate within families, but the comparison with the comparison with the standard remains valid under dependence. Last, using the frailty model concept, the multiplicative model, where the baseline hazards are known, is extended by adding a random frailty term that is based on the positive stable or the gamma distribution. Comparisons between the two frailty distributions are performed by simulation. Based on the results from various approaches, it is concluded that the siblings of centenarians had significant lower mortality rates as compared to their cohorts. The frailty models also indicate significant correlations between the life spans of the siblings. ^

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.

Transport of prion protein across the blood-brain barrier.

The cellular form of the prion protein (PrP(c)) is necessary for the development of prion diseases and is a highly conserved protein that may play a role in neuroprotection. PrP(c) is found in both blood and cerebrospinal fluid and is likely produced by both peripheral tissues and the central nervous system (CNS). Exchange of PrP(c) between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications, but it is unknown whether PrP(c) can cross the blood-brain barrier (BBB). Here, we found that radioactively labeled PrP(c) crossed the BBB in both the brain-to-blood and blood-to-brain directions. PrP(c) was enzymatically stable in blood and in brain, was cleared by liver and kidney, and was sequestered by spleen and the cervical lymph nodes. Circulating PrP(c) entered all regions of the CNS, but uptake by the lumbar and cervical spinal cord, hypothalamus, thalamus, and striatum was particularly high. These results show that PrP(c) has bidirectional, saturable transport across the BBB and selectively targets some CNS regions. Such transport may play a role in PrP(c) function and prion replication.

Postoperative renal function preservation with nonischemic femoral arterial cannulation for thoracoabdominal aortic repair.

BACKGROUND: Renal failure after thoracoabdominal aortic repair is a significant clinical problem. Distal aortic perfusion for organ and spinal cord protection requires cannulation of the left femoral artery. In 2006, we reported the finding that direct cannulation led to leg ischemia in some patients and was associated with increased renal failure. After this finding, we modified our perfusion technique to eliminate leg ischemia from cannulation. In this article, we present the effects of this change on postoperative renal function. METHODS: Between February 1991 and July 2008, we repaired 1464 thoracoabdominal aortic aneurysms. Distal aortic perfusion was used in 1088, and these were studied. Median patient age was 68 years, and 378 (35%) were women. In September 2006, we began to adopt a sidearm femoral cannulation technique that provides distal aortic perfusion while maintaining downstream flow to the leg. This was used in 167 patients (15%). We measured the joint effects of preoperative glomerular filtration rate (GFR) and cannulation technique on the highest postoperative creatinine level, postoperative renal failure, and death. Analysis was by multiple linear or logistic regression with interaction. RESULTS: The preoperative GFR was the strongest predictor of postoperative renal dysfunction and death. No significant main effects of sidearm cannulation were noted. For peak creatinine level and postoperative renal failure, however, strong interactions between preoperative GFR and sidearm cannulation were present, resulting in reductions of postoperative renal complications of 15% to 20% when GFR was <60 mL>/min/1.73 m(2). For normal GFR, the effect was negated or even reversed at very high levels of GFR. Mortality, although not significantly affected by sidearm cannulation, showed a similar trend to the renal outcomes. CONCLUSION: Use of sidearm cannulation is associated with a clinically important and highly statistically significant reduction in postoperative renal complications in patients with a low GFR. Reduced renal effect of skeletal muscle ischemia is the proposed mechanism. Effects among patients with good preoperative renal function are less clear. A randomized trial is needed.

Sulforaphane improves cognitive function administered following traumatic brain injury.

Recent studies have shown that sulforaphane, a naturally occurring compound that is found in cruciferous vegetables, offers cellular protection in several models of brain injury. When administered following traumatic brain injury (TBI), sulforaphane has been demonstrated to attenuate blood-brain barrier permeability and reduce cerebral edema. These beneficial effects of sulforaphane have been shown to involve induction of a group of cytoprotective, Nrf2-driven genes, whose protein products include free radical scavenging and detoxifying enzymes. However, the influence of sulforaphane on post-injury cognitive deficits has not been examined. In this study, we examined if sulforaphane, when administered following cortical impact injury, can improve the performance of rats tested in hippocampal- and prefrontal cortex-dependent tasks. Our results indicate that sulforaphane treatment improves performance in the Morris water maze task (as indicated by decreased latencies during learning and platform localization during a probe trial) and reduces working memory dysfunction (tested using the delayed match-to-place task). These behavioral improvements were only observed when the treatment was initiated 1h, but not 6h, post-injury. These studies support the use of sulforaphane in the treatment of TBI, and extend the previously observed protective effects to include enhanced cognition.

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Role of phosphatidylethanolamine in the function and assembly of phenylalanine-specific permease of Escherichia coli

Transmembrane segments of polytopic membrane proteins once inserted are generally considered stably oriented due to the large free energy barrier for topological reorientation of adjacent extra-membrane domains. However, proper topology and function of the polytopic membrane protein lactose permease (LacY) of Escherichia coli is dependent on the membrane phospholipid composition revealing topological dynamics of transmembrane domains (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107–2116). The high affinity phenylalanine permease PheP shares many topological similarities with LacY. In this study, mutant E. coli cells lacking phosphatidylethanolamine (PE) as a membrane component were used to evaluate the role of PE in the function and assembly of PheP. Active transport of phenylalanine by cells lacking PE was severely inhibited (both Vmax and Km were altered), whereas the PheP protein level in membranes was unaffected. Cysteine residues were introduced into predicted periplasmic or cytoplasmic segments of cysteine-less PheP, and the topology of the protein was explored using a membrane-impermeable thiol-specific biotinylated probe. Based on the biotinylation patterns of PheP in whole cells, the N-terminus and adjoining transmembrane hairpin of PheP adopted an inverted topological orientation in PE-lacking cells. Introduction of PE following the assembly of PheP triggered a reorientation of the N-terminus and adjacent hairpin to their native orientation associated with regain of wild type transport function. These results coupled with the results for LacY support a specific role for membrane lipid composition in determining topological organization and function of membrane proteins. Several other secondary symporters are compromised for activity in PE-lacking cells suggesting that lipid-assisted topogenesis is a general property of such transporters. The reversible orientation of these secondary transport proteins in response to a change of phospholipid composition might be a result of inherent conformational flexibility necessary for transport function or during protein assembly. ^

Validity and reliability of using MVP 4 Function Walk4Life digital pedometers to assess physical activity levels among preschool-aged Head Start children

The Surgeon General recommends preschoolers 3-5 years old accumulate 60 minutes of moderate-to-vigorous physical activity (MVPA) per day. However, there is limited data measuring physical activity (PA) and MVPA amongst this population. The purpose of this cross-sectional study is to determine the validity, reliability, and feasibility of using MVP 4 Function Walk4Life digital pedometers (MVP-4) in measuring MVPA among preschoolers using the newly modified direct observational technique, System for Observing Fitness Instruction Time-Preschool Version (SOFIT-P) as the gold standard. An ethnically diverse population of 3-5 year old underserved children were recruited from two Harris County Department of Education (HCDE) Head Start centers. For 2 days at baseline and 2 days at post-test, 75 children enrolled wore MVP-4 pedometers for approximately 6-hours per observation day and were observed using SOFIT-P during predominantly active times. Statistical analyses used Pearson "r" correlation coefficients to determine mean minutes of PA and MVPA, convergent and criterion validity, and reliability. Significance was set at p = <0.05. Feasibility was determined through process evaluation information collected during this study via observations from data collectors and teacher input. Results show mean minutes of PA and MVPA ranged between 30-42 and 11-14 minutes, respectively. Convergent validity comparing BMI percentiles with MVP-4 PA outcomes show no significance at pre-test; however, each measurement at post-test showed significance for MVPA (p = 0.0247, p = 0.0056), respectively. Criterion validity comparing percent MVPA time between SOFIT-P and MVP-4 pedometers was determined; however, results deemed insufficient due to inconsistency in observation times while using the newly developed SOFIT-P. Reliability measures show no significance at pre-test, yet show significant results for all PA outcomes at post-test (p = 0.001, p = 0.001, p = 0.0010, p = 0.003), respectively. Finally, MVP-4 pedometers lacked feasibility due to logistical barriers in design. Researchers feel the significant results at post-test are secondary to increased familiarity and more accurate placement of pedometers across time. Researchers suggest manufacturers of MVP-4 pedometers further modify the instrument for ease of use with this population, following which future studies ought to determine validity using objective measures or all-day direct observation techniques.^

A cross-sectional study of human symptomatology and residential formaldehyde concentrations

The cross-sectional study was performed to quantify the prevalence of symtomatology in residents of mobile homes as a function of indoor formaldehyde concentration. Formaldehyde concentrations were monitored for a seven hour period with an automated wet-chemical colorimetric analyzer. The health status of family members was ascertained by administration of questionnaires and physical exams. This is the first investigation to perform clinical assessments on residents undergoing concurrent exposure assessment in the home.^ Only 22.8% of households eligible for participation chose to cooperate. Monitoring data and health evaluations were obtained from 155 households in four Texas counties. A total of 428 residents (86.1%) were available for examination during the sampling hours. The study population included 45 infants, 126 children, and 257 adults.^ Formaldehyde concentration was not found to be significantly associated with increased risks for symptoms and signs of ocular irritation, dermal anomalies, or malaise. Three associations were identified that warrant further investigation. The relative odds associated with a doubling of formaldehyde concentration was significantly associated with parenchymal rales in adults and children. However, risk was modified by log respirable suspended particulate concentrations. Due to the presence of modification by a continuous variable, prevalence odds ratios (POR) and 95% confidence intervals (95% CI) for these associations are presented in tables. A doubling of formaldehyde concentration was also associated with an increased risk of perceived tightness in the chest in adults. Prevalence odds ratios are presented in a table due to effect modification by the average number of hours spent indoors on weekdays. Furthermore, a doubling of formaldehyde concentration was associated with an increased risk of drowsiness in children (POR = 2.60; 95% CI 1.04-6.51) and adults (POR = 1.94; 95% CI 1.20-3.14). ^

Effect of patient accrual patterns on power and size of log-rank test for time-to-event outcome in clinical trials

The determination of size as well as power of a test is a vital part of a Clinical Trial Design. This research focuses on the simulation of clinical trial data with time-to-event as the primary outcome. It investigates the impact of different recruitment patterns, and time dependent hazard structures on size and power of the log-rank test. A non-homogeneous Poisson process is used to simulate entry times according to the different accrual patterns. A Weibull distribution is employed to simulate survival times according to the different hazard structures. The current study utilizes simulation methods to evaluate the effect of different recruitment patterns on size and power estimates of the log-rank test. The size of the log-rank test is estimated by simulating survival times with identical hazard rates between the treatment and the control arm of the study resulting in a hazard ratio of one. Powers of the log-rank test at specific values of hazard ratio (≠1) are estimated by simulating survival times with different, but proportional hazard rates for the two arms of the study. Different shapes (constant, decreasing, or increasing) of the hazard function of the Weibull distribution are also considered to assess the effect of hazard structure on the size and power of the log-rank test. ^