Multimodal Regulation Orchestrates Normal and Complex Disease States in the Retina.

Regulation of biological processes occurs through complex, synergistic mechanisms. In this study, we discovered the synergistic orchestration of multiple mechanisms regulating the normal and diseased state (age related macular degeneration, AMD) in the retina. We uncovered gene networks with overlapping feedback loops that are modulated by nuclear hormone receptors (NHR), miRNAs, and epigenetic factors. We utilized a comprehensive filtering and pathway analysis strategy comparing miRNA and microarray data between three mouse models and human donor eyes (normal and AMD). The mouse models lack key NHRS (Nr2e3, RORA) or epigenetic (Ezh2) factors. Fifty-four total miRNAs were differentially expressed, potentially targeting over 150 genes in 18 major representative networks including angiogenesis, metabolism, and immunity. We identified sixty-eight genes and 5 miRNAS directly regulated by NR2E3 and/or RORA. After a comprehensive analysis, we discovered multimodal regulation by miRNA, NHRs, and epigenetic factors of three miRNAs (miR-466, miR1187, and miR-710) and two genes (Ell2 and Entpd1) that are also associated with AMD. These studies provide insight into the complex, dynamic modulation of gene networks as well as their impact on human disease, and provide novel data for the development of innovative and more effective therapeutics.

Studying the peripheral sympathetic nervous system and neuroblastoma in zebrafish.

The zebrafish serves as an excellent model to study vertebrate development and disease. Optically clear embryos, combined with tissue-specific fluorescent reporters, permit direct visualization and measurement of peripheral nervous system formation in real time. Additionally, the model is amenable to rapid cellular, molecular, and genetic approaches to determine how developmental mechanisms contribute to disease states, such as cancer. In this chapter, we describe the development of the peripheral sympathetic nervous system (PSNS) in general, and our current understanding of genetic pathways important in zebrafish PSNS development specifically. We also illustrate how zebrafish genetics is used to identify new mechanisms controlling PSNS development and methods for interrogating the potential role of PSNS developmental pathways in neuroblastoma pathogenesis in vivo using the zebrafish MYCN-driven neuroblastoma model.

Acute gastrointestinal illness following a prolonged community-wide water emergency.

The drinking water infrastructure in the United States is ageing; extreme weather events place additional stress on water systems that can lead to interruptions in the delivery of safe drinking water. We investigated the association between household exposures to water service problems and acute gastrointestinal illness (AGI) and acute respiratory illness (ARI) in Alabama communities that experienced a freeze-related community-wide water emergency. Following the water emergency, investigators conducted a household survey. Logistic regression models were used to estimate adjusted prevalence ratios (aPR) and 95% confidence intervals (CI) for self-reported AGI and ARI by water exposures. AGI was higher in households that lost water service for ⩾7 days (aPR 2·4, 95% CI 1·1-5·2) and experienced low water pressure for ⩾7 days (aPR 3·6, 95% CI 1·4-9·0) compared to households that experienced normal service and pressure; prevalence of AGI increased with increasing duration of water service interruptions. Investments in the ageing drinking water infrastructure are needed to prevent future low-pressure events and to maintain uninterrupted access to the fundamental public health protection provided by safe water supplies. Households and communities need to increase their awareness of and preparedness for water emergencies to mitigate adverse health impacts.

Seasonal and dose-dependent effects of intracerebroventricular leptin on lh secretion and appetite in sheep.

The role of leptin in neuroendocrine appetite and reproductive regulation remains to be fully resolved. A series of three experiments was conducted using adequately nourished oestradiol-implanted castrated male sheep. In a cross-over design (n=6), responses to a single i.c.v. (third ventricle) injection of leptin (0.5, 1.0 and 1.5 mg ovine leptin (oLEP) and 1.0 mg murine leptin (mLEP)), N-methyl-D-aspartate (NMDA, 20 micro g) or 0.9% saline (control) were measured in terms of LH secretion (4 h post-injection compared with 4 h pre-injection) and appetite (during 2 h post-injection) in autumn (Experiment 1). NMDA and 1.0 mg oLEP treatments were repeated in the same sheep in the following spring (Experiment 2). With an additional 12 sheep (n=18 in cross-over design), responses to low-dose 'physiological' i.c.v. infusion of leptin (8 ng/h for 12 h daily for 4 days), insulin (0.7 ng/h) and artificial cerebrospinal fluid were measured in the next spring (Experiment 3). LH was studied over 8 h and appetite over 1 h on days 1 and 4 of infusion. In Experiment 1 (autumn), oLEP overall increased LH pulse frequency by up to 110% (P<0.05), decreased LH pulse amplitude (P<0.05) and decreased appetite (P<0.05). mLEP reduced LH pulse amplitude (P<0.05) without significant effect on appetite, while NMDA reduced appetite (P<0.05) but had no effect on LH. In Experiment 2 (spring), LH responses were 'surge-like' with highly significant increases in the moving average LH concentration after 1.0 mg oLEP (P<0.001) and after NMDA (P<0.001). Compared with similar analysis of experiment 1 results, the LH response in spring was greater than that in autumn for both 1.0 mg oLEP (P<0.05) and NMDA (P<0.005). Conversely, unlike in autumn (Experiment 1), there was no effect of 1.0 mg oLEP or NMDA on appetite in the spring (Experiment 2). In Experiment 3 (spring), 'physiological' i.c.v. infusion of oLEP or insulin increased LH pulse frequency by up to 100% (P<0.001) compared with the control infusion on both days 1 and 4, but there were no effects on appetite. These results indicate that intracerebral leptin both stimulates reproductive neuroendocrine output and decreases appetite in adequately nourished sheep. However, the responses of these two axes were dose-dependent and differentially affected by the time of year, suggesting dissociation of the neural pathways involved.

Filtration artifacts caused by overloading membrane filters.

The conventional practice of using 0.45 or 0.40 microm membranes to distinguish between the particulate and dissolved phases in natural waters neglects the importance of colloids. Many of the colloids in natural waters pass through 0.45 or 0.40 microm membranes, but a significant fraction at the upper end of the colloidal particle size range is retained. Membrane clogging during filtration decreases the effective pore size and can cause the retention of increasing amounts of colloids. This filtration artifact can cause serious errors in sampling and in assigning trace metals to various particle size classes. We evaluated the effect of membrane loading for two common membrane types (0.45 microm Millipore Durapore and 0.40 microm Nuclepore) on the retention of colloidal Fe, Al, Mn, and OM in three Connecticut rivers. In addition, we used a 1.0 microm Nuclepore membrane to estimate the amount of colloids in the 0.40-1.0 microm size fraction that are retained by membranes during conventional filtration. All samples were collected with clean techniques, and all filtrations were carried out in a class 100 clean room. A peristaltic pump, set at an initial flow rate of 120 mL/min, was used to pump samples through 47 mm diameter inline Teflon filter holders. Back pressure and flow rate were monitored during filtration, and both are good indicators for the onset of membrane clogging. The results show a consistent correlation between increasing back pressure and decreasing concentration of colloidal Fe and sometimes Al, Mn, and OM in the filtrate for all membrane types. Although the shape of the loading-retention curves varied dramatically by site and by membrane type, the essential relationship between back pressure, flow rate, and filtration artifacts during membrane clogging remained the same.

Pyruvate ingestion for 7 days does not improve aerobic performance in well-trained individuals.

The purposes of the present studies were to test the hypotheses that lower dosages of oral pyruvate ingestion would increase blood pyruvate concentration and that the ingestion of a commonly recommended dosage of pyruvate (7 g) for 7 days would enhance performance during intense aerobic exercise in well-trained individuals. Nine recreationally active subjects (8 women, 1 man) consumed 7, 15, and 25 g of pyruvate and were monitored for a 4-h period to determine whether blood metabolites were altered. Pyruvate consumption failed to significantly elevate blood pyruvate, and it had no effect on indexes of carbohydrate (blood glucose, lactate) or lipid metabolism (blood glycerol, plasma free fatty acids). As a follow-up, we administered 7 g/day of either placebo or pyruvate, for a 1-wk period to seven, well-trained male cyclists (maximal oxygen consumption, 62.3 +/- 3.0 ml. kg(-1). min(-1)) in a randomized, double-blind, crossover trial. Subjects cycled at 74-80% of their maximal oxygen consumption until exhaustion. There was no difference in performance times between the two trials (placebo, 91 +/- 9 min; pyruvate, 88 +/- 8 min). Measured blood parameters (insulin, peptide C, glucose, lactate, glycerol, free fatty acids) were also unaffected. Our results indicate that oral pyruvate supplementation does not increase blood pyruvate content and does not enhance performance during intense exercise in well-trained cyclists.

Comparison of serum and salivary cotinine measurements by a sensitive high-performance liquid chromatography-tandem mass spectrometry method as an indicator of exposure to tobacco smoke among smokers and nonsmokers.

Exposure to tobacco smoke, both from active smoking and from passive exposure to environmental tobacco smoke, can be monitored by measuring cotinine, a metabolite of nicotine, in a variety of biological sources including blood, urine, and saliva. Previously, a sensitive atmospheric-pressure ionization, tandem mass spectrometric (LC-API-MS-MS) method for cotinine measurements in serum was developed in support of a large, recurrent national epidemiologic investigation. The current study examined the application of this LC-API-MS-MS method to both serum and saliva cotinine measurements in a group of 200 healthy adults, including both smokers and nonsmokers. The primary objective of this study was to evaluate the relationship between serum and saliva cotinine concentrations to facilitate the linking of results from epidemiologic studies using salivary cotinine measurements to existing national data based on serum cotinine analyses. The results indicate that a simple, linear relationship can be developed to describe serum and saliva cotinine concentrations in an individual, and the expression describing this relationship can be used to estimate with reasonable accuracy (approximately +/- 10%) the serum cotinine concentration in an individual given his or her salivary cotinine result. It was further confirmed that saliva cotinine samples are generally quite stable during storage after collection, even at ambient temperatures, and this sample matrix appears to be well-suited to the requirements of many epidemiologic investigations.

The psychometric properties of the Homonegativity Scale.

This research details the psychometric properties of an instrument designed to measure negative attitudes toward gay men and lesbians (the Homonegativity Scale-HS). Four studies were conducted using adolescents (N = 1078), university students (N = 343), and members of the general population (N = 146). Results suggest that the HS possesses a unidimensional factor structure and a high degree of internal consistency for both male and female participants. As predicted, scores on the HS were positively correlated with machismo, authoritarianism, political conservatism, religiosity, and modern sexism. In addition, responses on the HS did not appear to be contaminated by social desirability bias. The usefulness of this measure in survey research is discussed.

At war in the fields of play: current perspectives on the nature and treatment of adolescent chemical dependency.

While the numbers of adolescents entering drug abuse treatment are not increasing dramatically, those entering treatment are getting sicker, according to clinical reports. Adolescents are entering treatment with multiple problems, including severe learning disorders, borderline personality disorders, multiple diagnoses of addiction, mental health and physical problems (including HIV/AIDS), and issues arising from child abuse and incest. A case study in client-therapist interaction is presented, highlighting the concept of the wounded healer. The disease model of addiction is discussed as it applies to adolescents, as is their need for habilitation, not rehabilitation. Current abuse trends and the drugs involved are also discussed. Multiple diagnoses, physiologic disorders, HIV testing and counseling, surgical problems, and maternal/neonatal complications are reviewed.

Addiction in adolescents.

Some symptoms seen in adolescents with the disease of chemical dependence are similar to those seen in adults. Because of their age, lack of personality development, dependent family role, immaturity, and acting out of age-related behavioral tendencies, however, symptoms specific to this population occur. These may become exacerbated and telescope--intensify and shorten--the progression of the disease. A plan to solve the problem of adolescent chemical dependence must focus on education, demonstration, cooperation, prevention, intervention, habilitation, treatment, and recovery. The phenomenon of denial in a chemically dependent adolescent yields a more complex delusional system that dictates age-specific intervention approaches. Habilitation is necessary for successful adolescent treatment and recovery because what is needed is an initial process of learning, not relearning or rehabilitation. If specific adolescent issues are addressed through comprehensive, multimodality treatment approaches, then treatment and recovery outcomes for chemically dependent adolescents and their families are substantially improved. Primary care physicians must be alert to the possibility of drug use in their young patients and aware of treatment options.

Psychiatric issues of adolescent chemical dependence.

Pediatricians, as well as other health care professionals, must always consider psychoactive drug use by adolescents as possible causes of general health or emotional problems, such as poor appetite, insomnia, problem behavior, academic underachievement, and loss of energy and motivation. Early diagnosis and intervention with youth can result in enormous benefits for the lives of the youth, families, and society. The physician must be alert to the adolescent who manifests such signs of abuse. This article details the recognition and management of these problems.

Identification of ninhydrin-positive caprolactam metabolites in the rat.

Caprolactam (2-oxohexamethylenimine) is metabolized in the rat partly to 4-hydroxycaprolactam or the corresponding free acid. The 4-hydroxy metabolite rearranges spontaneously in acidic aqueous medium to an equilibrium mixture of which 6-amino-gamma-caprolactone is the major component and 6-amino-4-hydroxyhexanoic acid is a minor component. Approximately 16% of the caprolactam ingested daily by rats fed a diet containing 3% caprolactam was excreted as the 4-hydroxy metabolite and a small amount was excreted as the non-hydroxylated acid, 6-aminohexanoic acid. The metabolites were isolated by ion-exchange chromatography and identified by gas chromatography-mass spectroscopy and infra-red and nuclear magnetic resonance spectroscopy.

Comparative biochemistry and fine structure of atrial and ventricular myocardium during autolysis in vitro.

In a previous study we found that the development of fine structural alteration in atrial myocardium made ischaemic in vivo was slower than has been observed for ventricular myocardium. To explore possible reasons for this, parallel samples of atrial (A) and ventricular (V) myocardium undergoing autolysis (ischaemic necrosis) in vitro at 37 degrees C were studied for up to 2 hours. At 15-minute intervals tissue was snap-frozen for measurement of pH, lactate, and adenine metabolites by HPLC. In half the experiments comparable specimens were taken for electron microscopic examination as well. Fine structural alteration developed less uniformly and more slowly in A than in V. The most striking metabolic differences between A and V were: A had a consistently higher tissue pH and lower lactate level The sum of the adenine + hypoxanthine metabolites was essentially constant but significantly different for each (A = 5.04 +/- 0.12 (s.e.m.), V = 7.71 +/- 0.15 (s.e.m.) mumol/g wet tissue weight) Initial ATP levels were lower (40% less) in A The maximum accumulation of AMP was higher in A, despite its smaller pool of adenine metabolites Both adenosine and inosine showed slower rates of change in A. These results suggest that during early, severe ischaemic injury A and V show differing activities of 5'-nucleotidase.

The potassium/sodium ratio in the demonstration of sudden ischemic cardiac death: a critical appraisal.

The practical value of measuring the ratio of potassium ion (K+) to sodium ion (Na+) in myocardium as an indicator of early inapparent infarction in sudden cardiac death was assessed using a series of 29 human hearts from selected coroner's autopsies together with experimental material from dogs, including infarcts of 5 min to 4 h duration. Samples for electrolyte analysis were derived from a transverse slice of each heart, taken through both ventricles midway between base and apex, all slices being completely subdivided into a numbered sequence of blocks. Ratios were mapped and compared with macroscopic enzyme staining and histological stains for injured muscle. Detailed examination of coronary arteries was performed on all human cases. Measurement of the K+/Na+ ratio did not detect all human cases of proven acute coronary occlusion and did not unequivocally demonstrate experimental infarcts less than 2 h old. Moreover, all ratios fell with increasing duration of autolysis, emphasizing the need for multiple sampling so that each heart may serve as its own control. As a routine test, therefore, the method is both impracticable and unreliable and as previously used has been subject to misinterpretation.

Effects of proton release from adenine nucleotide degradation during ischemic necrosis of myocardium in vitro.

The effects of the progressive production of hydrogen ions and inorganic phosphate from adenine nucleotides on the lowering of pH in heart muscle has been investigated in dog myocardium maintained under anoxic conditions in vitro at 37 degrees C. Tissue samples were taken at 15-min intervals for up to 105 min and the following biochemical parameters determined: pH, lactate, Pi, ATP, ADP, AMP, G6P, and PC in selected instances. From these data the net proton changes during prolonged anoxia were calculated, assuming homogeneity of the tissue milieu. Net proton change was negative after 15 min and became increasingly more so throughout the remainder of the experimental period, indicating that adenine nucleotide catabolism in fact has a protective effect against fall in pH. When tissue pH falls to ca. 6.2 (45-60 min anoxia), proton production due to lactic acid is reduced by approximately 16% because of absorption of protons by phosphate and ammonia liberated from the nucleotides.