A Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms.

Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes.

Evaluating systems of care for children: utility of the clinical case conference.

A clinical assessment of the Mental Health Services Program for Youth, a national initiative to integrate systems and coordinate care for severely emotionally disturbed children, was designed to augment and enrich the larger evaluation of program structure. Case conferences were used as a method of examining the effects of collaborative systems of care on vulnerable individuals and of generating clinical insight and understanding. Case vignettes are presented and discussed in terms of the contributions and shortcomings of current system-of-care efforts.

Bridging the gap between service need and service utilization: a school-based mental health program.

In an effort to bridge the gap between service need and service utilization, an urban based, university affiliated children's psychiatric outpatient clinic has implemented a program which provides mental health services in inner city schools. When compared with the central clinic populations (N = 304), the school sample (N = 44) was markedly socioeconomically disadvantaged, minority, and as psychiatrically impaired as the central clinic population. School based mental health services have the potential for bridging the gap between need and utilization by reaching disadvantaged children who would otherwise not have access to these services. Implications for such services are discussed.

Obsessive-compulsive characteristics: from symptoms to syndrome.

OBJECTIVE: To assess the distribution and severity of obsessions and compulsions in a nonclinical adolescent population. METHOD: During preinduction military screening, 861 sixteen-year-old Israelis completed a questionnaire regarding the lifetime presence of eight obsessive-compulsive (OC) symptoms and three severity measures. The presence or absence of obsessive-compulsive disorder (OCD) or subclinical OCD was ascertained by an independent interview. RESULTS: Although only 8.0% and 6.3% of respondents reported disturbing and intrusive thoughts, respectively, 27% to 72% of subjects endorsed the six remaining OCD symptoms. Twenty percent of subjects regarded the symptoms they endorsed as senseless and 3.5% found them disturbing; 8% reported spending more than an hour daily on symptoms. OCD and subclinical OCD cases differed significantly from non-OCD cases, but not from each other, in distress and mean number of symptoms. Although the distribution of nine of the items differed for noncases, compared with OCD and subclinical OCD cases, the distributions for all items overlapped markedly across the three groups. CONCLUSIONS: OC phenomena appear to be on a continuum with few symptoms and minimal severity at one end and many symptoms and severe impairment on the other. Defining optimal cutoff points for distinguishing between psychiatric disorder and OC phenomena that are common in the general population remains an open question.

Determinants of reliability in psychiatric surveys of children aged 6-12.

The reliability of young children's self reports of psychiatric information is a concern of epidemiologists and clinicians alike. This paper explores the determinants of test-retest reliability in a sample of children from the general population using reliability coefficients constructed from a kappa statistic. Age, cognitive ability, and gender are related to consistency of reports in a test-retest paradigm. Controlling for age, cognitive ability and gender, children report more reliably on observable behaviors, and less reliably on questions involving unspecified time, reflections of one's own thoughts, and comparison of themselves with others. The reliability of reports of emotions lies between these two extremes. Surprisingly, sentence length of up to 40 words and psychiatric impairment of the child as measured by the Child Global Assessment Scale did not influence reliability. As might be expected, parents' reports of their children are more reliable than their children's reports.

Clinical, sociodemographic, and systems risk factors for attrition in a children's mental health clinic.

Clinical, sociodemographic, and systems risk factors for attrition at a child guidance clinic were identified. All patients who completed the intake process were followed through various clinic phases. Lower socioeconomic status and low family cohesion predicted dropout. Dropouts and continuers were equally psychiatrically impaired. The Family Environment Scale was the only standardized measure that distinguished between dropouts and continuers.

Reliability of diagnostic reporting for children aged 6-11 years: a test-retest study of the Diagnostic Interview Schedule for Children-Revised.

OBJECTIVE: This study examined the reliability of symptom reporting by community children of elementary school age and their parents on a version of the Diagnostic Interview Schedule for Children-Revised (DISC-R). METHOD: A sample of 109 children aged 6-11 years from an ongoing epidemiologic study were recruited for retest DISC-R interviews after completion of the study protocol. Retest interviews took place 7-18 days after the first interview and were conducted by interviewers who had no prior information about the subjects. Test-retest reliability for five common childhood psychiatric diagnoses was evaluated with the kappa statistic; the intraclass correlation coefficient was used to evaluate test-retest reliability of symptom scales. RESULTS: The reliability of the parents' reports on the DISC-R was good to excellent for attention deficit hyperactivity disorder and separation anxiety disorder; it was fair for overanxious disorder, oppositional defiant disorder, and conduct disorder. The children reported many fewer symptoms than the parents except for separation anxiety disorder; reliability was fair for separation anxiety disorder and poor for attention deficit hyperactivity disorder. The children were particularly unreliable in reporting about time factors, such as duration and onset of symptoms. When symptoms were considered without duration and onset, children's reports reached fair reliability for separation anxiety disorder, overanxious disorder, and attention deficit hyperactivity disorder but remained poor for oppositional defiant disorder. CONCLUSIONS: The results suggest that highly structured diagnostic interviews such as the DISC-R may not be appropriate for use with younger children of elementary school age in community-based studies.

Nurses' attitudes toward homeless women: a barrier to change.

Close to a third of the almost 4 million homeless persons in the United States are women. They have multiple physical and mental health problems directly attributable to their homelessness. Studies have indicated that their failure to seek health care is partially a function of the barrier created by negative attitudes of nurses toward homeless women. Strategies for changing nurses' attitudes, built on attitude change theory, include methods for increasing knowledge and experience in the treatment of homeless women.

Congenital hypertrophy of retinal pigment epithelium in patients with colonic polyps associated with cancer family syndrome.

Congenital hypertrophy of retinal pigment epithelium (CHRPE) has been shown to be a frequent extracolonic manifestation of adenomatous polyposis coli (APC). The presence of CHRPE in patients with adenomatous polyps from families with cancer family syndrome suggests possible involvement of the APC gene locus in syndromes associated with less florid polyp formation than seen in APC. It also emphasises that caution must be exercised in using the presence of CHRPE clinically as a marker for APC in isolated at-risk individuals.

Cloning and characterization of a gene encoding pig epidermal growth factor.

A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized. The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence. In addition, conservation of the intron-exon boundaries between the two species was generally observed. Although the pig exon 21 appeared to lack a single nucleotide at its 5' end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5' end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact. Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity. A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast. The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.

Oxidative effects of laser photocoagulation.

Diabetic proliferative retinopathy is a common and sight-threatening condition. Oxidative stress is an integral and possibly causative part of the pathogenesis. Although laser photocoagulation is usually a beneficial treatment it remains unclear how it works. The possibility that it induces a sudden, temporary increase in free radical activity either by direct thermal damage or by oxygen reperfusion is explored in this clinical study by measuring the oxidative status in the peripheral blood of 13 patients undergoing panretinal photocoagulation. There were significant increases at one hour in malondialdehyde-like material (MDA-LM), 8.1 (6.9-9.6) nmol/mL, to 9.1 (7.6-9.8) nmol/mL, (less than 0.005); plasma thiols (PSH), 423 (352-457) microns/L, to 444 (382-478) microns/L, (p less than 0.005) and red cell reduced glutathione (GSH), 1357 (1295-1655) microns/L, to 1480 (1305-1760) microns/L, (p less than 0.01). Diene conjugates rose over the first hour 0.55 (0.36-0.79) od/mL, to 0.58 (0.34-0.85) od/mL falling to 0.56 (0.36-0.79) od/mL at 2 h but these changes were not significant. At 2 h, MDA-LM 8.4 (6.7-9.6) nmol/mL and PSH 404 (379-462) microns/L had returned to baseline but GSH remained significantly elevated 1500 (1325-1675) microns/L, (p less than 0.005 compared to baseline). This is a new observation and in some circumstances such generation of free radicals could explain the mechanism behind the complications of photocoagulation by direct or indirect damage to vascular endothelium leading to increased vascular permeability manifest as macular oedema or choroidal effusions.

Long-term follow-up of retinal blood flow in diabetes using the blue light entoptic phenomenon.

The blue light entopic phenomenon was used to measure retinal blood flow in 87 diabetics and 10 normal controls in a longitudinal study. The time interval between the initial and final studies was 21-39 months (mean 31.4). The retinal blood flow velocity did not change in the 10 subjects in the control group nor in those nine patients with no retinopathy, all but one of whom remained free of retinal lesions. In the 24 with background retinopathy the flow velocity fell significantly from 0.71 (SD 0.35) mm/s to 0.48 (0.13) mm/s (p less than 0.01). The velocity also decreased in the preproliferative group of 16 patients from 0.62 (0.39) mm/s to 0.41 (0.14) mm/s (p less than 0.05). The retinopathy status changed in 11 of this group. In those with proliferative retinopathy (now treated) and those treated previously there was no change in flow velocity.

Respiratory effect on the blood volume of pulmonary capillaries.

We measured the density variations of aortic blood from rabbits ventilated by a positive end inspiratory pressure of 6 mmHg or a negative box pressure of the same magnitude. When calculated from the density variations, the fluctuations in blood volume of the pulmonary capillaries within one cycle as induced by an intermittent positive pressure ventilation were found to be similar to the ones induced by an intermittent negative pressure ventilation. Using these volumetric fluctuations as a means to assess the transpulmonary pressure and the transmural pressure across the pulmonary capillaries, we conclude that the switching of the ventilation method did not alter the cyclic fluctuations of these pressures.

Plasma adenosine and hypoxemia in patients with sleep apnea.

Severe hypoxemia causes ATP depletion and increased adenosine production in many body tissues. Therefore we hypothesized that patients with sleep apnea and severe hypoxemia during sleep have higher adenosine production and higher plasma adenosine levels than patients without hypoxemia. Twelve patients with sleep apnea and six normal volunteers had plasma adenosine levels measured by high-performance liquid chromatography. Each patient with sleep apnea had a polysomnograph sleep study with oxyhemoglobin saturation continuously recorded. Five of 12 patients with sleep apnea had both sleep apnea and severe hypoxemia during sleep. These patients with severe nocturnal hypoxemia had significantly higher plasma adenosine levels (means +/- SD 9.7 +/- 5.5 X 10(-8) M) than either a group of six normal volunteers (3.5 +/- 0.7 X 10(-8) M) or a group of seven patients with sleep apnea without hypoxemia at night (3.1 +/- 1.5 X 10(-8) M) (P less than 0.01). In addition plasma adenosine levels were significantly correlated with two indexes of nocturnal hypoxemia (desaturation index rs = 0.79, and median oxyhemoglobin saturation during sleep rs = -0.75, P less than 0.01). Plasma adenosine markedly fell to a normal level in the only two patients with sleep apnea who had successful treatment of their multiple apneas and accompanying severe hypoxemia during sleep.

Autoregulation of retinal blood flow in diabetic retinopathy measured by the blue-light entoptic technique.

Autoregulation of retinal blood flow was measured in 23 diabetic patients, 9 with nonproliferative retinopathy, 9 with proliferative retinopathy, and 5 with photocoagulated retinopathy. Three of the proliferative patients were studied on two occasions, at the proliferative stage and after photocoagulation. The stimuli to autoregulation used were isocapnic hyperoxia and hypoxia. Flow velocity of leukocytes in the perifoveal circulation was measured using the blue-field entoptic technique, and retinal artery and vein diameters were measured using a computerized digitizing system. Results under conditions of isocapnic hypoxia showed a mean increase in flow velocity of 41.0 +/- 33.0% (P less than 0.05) in nonproliferative retinopathy, 3.5 +/- 17.0% (not significant) in proliferative retinopathy, and 30.0 +/- 17.0% (P less than 0.05) after photocoagulation. In hyperoxia, flow velocity fell by a mean of 21.0 +/- 21.6% (P less than 0.05) in nonproliferative retinopathy, 33.0 +/- 19.9% (P less than 0.005) in proliferative retinopathy, and 21.0 +/- 26.0% (not significant) in photocoagulated retinopathy. The results are compared with a group of nondiabetic subjects reported previously.

The effect of acute hyperglycemia on flow velocity in the macular capillaries.

The effect of acute hyperglycemia on retinal blood flow was measured in 12 diabetic patients (mean blood glucose, 276 mg%) on continuous subcutaneous insulin infusion and six nondiabetic controls (mean blood glucose, 198 mg%). Flow velocity measurements in macular capillaries were made using the blue field entoptic method. Retinal artery and vein diameters were measured using red-free fundus photographs. No significant change in flow velocity or retinal vessel diameter was noted in either group.

Long-term follow-up of patients who underwent yttrium-90 pituitary implantation for treatment of proliferative diabetic retinopathy.

Between 1960 and 1976 117 patients underwent pituitary implantation with yttrium-90 (90Y) for treatment of proliferative retinopathy at the Hammersmith Hospital, London. Mean age at operation was 35 +/- 11 years (mean +/- SD), and mean duration of diabetes 18.6 +/- 10.0 years. Mean insulin dosage prior to implant was 67.2 +/- 24 units, falling to 30.4 +/- 14.9 units post-implant. Thirty-two per cent of patients are still living, 60% are deceased and 8% are lost to follow-up. The 5-year survival rate was 82%. Of the causes of death, 21% died of infection, adrenal insufficiency or hypoglycaemia, 12% of renal failure, and 47% of myocardial or cerebral vascular disease. Ophthalmological follow-up was carried out on the 100 patients operated on between 1965 and 1976. The mean age of this group at implant was 35 +/- 10.5 years, and mean duration of diabetes 17.2 +/- 8.7 years. Visual acuity in the better eye at operation was 6/12 or better in 84% of patients, and this percentage remained similar at the time of the 5 and 10 year follow-up. Blindness (6/60 or worse) in both eyes was present in 12% of patients at the time of 5 and 10 year assessments. By 5 years new vessels on the disc had improved from a mean grading of 2.7 +/- 1.6 to 0.8 +/- 1.2 (p less than 0.001), and by 10 years there was no disc neovascularisation in any eye. There was a similar improvement in the grading of hard exudates, microaneurysms and haemorrhages, but there was an increase in fibrous retinitis proliferans.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of fluorescein glucuronide and its metabolism in vitreous fluorophotometry.

Rabbits were given fluorescein or fluorescein glucuronide intravenously. Fluorescein and fluorescein glucuronide concentrations in plasma and vitreous samples were measured by high-performance liquid chromatography. Vitreous fluorophotometry was performed using the Fluorotron Master to compare scans after administration of fluorescein and fluorescein glucuronide, and for comparison of in vivo fluorescence with in vitro high-performance liquid chromatography analysis. Fluorescein glucuronide was shown to enter the vitreous as early as 1 hr after injection. Fluorescein glucuronide was the dominant molecule in both vitreous and plasma of all rabbits at 6 hr. Because fluorescein glucuronide has a lower fluorescence than fluorescein, the fluorophotometer overestimates the vitreous concentration of fluorescein after its administration. Since fluorescein is metabolized rapidly to fluorescein glucuronide in man, entry of fluorescein glucuronide into the eye should be considered in measurements of blood-ocular barrier permeability by vitreous fluorophotometry.