Application of the systematic "DAmP" approach to create a partially defective C. albicans mutant.

An understanding of gene function often relies upon creating multiple kinds of alleles. Functional analysis in Candida albicans, a major fungal pathogen, has generally included characterization of mutant strains with insertion or deletion alleles and over-expression alleles. Here we use in C. albicans another type of allele that has been employed effectively in the model yeast Saccharomyces cerevisiae, a "Decreased Abundance by mRNA Perturbation" (DAmP) allele (Yan et al., 2008). DAmP alleles are created systematically through replacement of 30 noncoding regions with nonfunctional heterologous sequences, and thus are broadly applicable. We used a DAmP allele to probe the function of Sun41, a surface protein with roles in cell wall integrity, cell-cell adherence, hyphal formation, and biofilm formation that has been suggested as a possible therapeutic target (Firon et al., 2007; Hiller et al., 2007; Norice et al., 2007). A SUN41-DAmP allele results in approximately 10-fold reduced levels of SUN41 RNA, and yields intermediate phenotypes in most assays. We report that a sun41Δ/Δ mutant is defective in biofilm formation in vivo, and that the SUN41-DAmP allele complements that defect. This finding argues that Sun41 may not be an ideal therapeutic target for biofilm inhibition, since a 90% decrease in activity has little effect on biofilm formation in vivo. We anticipate that DAmP alleles of C. albicans genes will be informative for analysis of other prospective drug targets, including essential genes.

Unintended changes in cognition, mood, and behavior arising from cell-based interventions for neurological conditions: ethical challenges.

The prospect of using cell-based interventions (CBIs) to treat neurological conditions raises several important ethical and policy questions. In this target article, we focus on issues related to the unique constellation of traits that characterize CBIs targeted at the central nervous system. In particular, there is at least a theoretical prospect that these cells will alter the recipients' cognition, mood, and behavior-brain functions that are central to our concept of the self. The potential for such changes, although perhaps remote, is cause for concern and careful ethical analysis. Both to enable better informed consent in the future and as an end in itself, we argue that early human trials of CBIs for neurological conditions must monitor subjects for changes in cognition, mood, and behavior; further, we recommend concrete steps for that monitoring. Such steps will help better characterize the potential risks and benefits of CBIs as they are tested and potentially used for treatment.

Pupillometric assessment of dysautonomia in pediatric bowel and bladder dysfunction: a pilot study.

Bowel and bladder dysfunction (BBD) refers to a heterogeneous group of voiding disorders, accounting for an estimated 40% of pediatric urology visits. Symptoms of BBD include enuresis, urgency, and urinary retention, often accompanied by constipation. The aim of this pilot study was to explore whether a pupillary response can be characterized for BBD, by examining the pupillary light reflex (PLR) before and after voiding among patients with BBD. A total of 28 patients aged from 7 to 21 years were recruited from the Wetting, Infections, and Stooling Help clinic at Children's National Medical Center. An infrared pupilometer was used to assess the PLR. Both baseline static and dynamic pupillometry assessments were obtained before and after voiding. Measurements were also taken after 5 min in the supine position, followed by 5 min standing to induce an orthostatic stressor. Visual inspection of the graphed data revealed a characteristic shape in 11 of 28 patients with voiding symptoms. In these 11 patients, the redilation arm of the PLR shows a 'notch,' or a brief reconstriction of the pupil before resting pupil size is reestablished (figure). This feature of the PLR has not been seen in previous and parallel studies using pupillometry to evaluate other populations. The results of this study suggest that a subset of patients with BBD may have a significant perturbation of autonomic regulation, identifiable through analysis of the PLR. To our knowledge, this 'notch' during redilation has not been previously described or seen in other patient populations and may represent a distinctive and readily identifiable physiologic marker of disease. These results are broadly aligned with results of other studies that have examined ANS activity in patients with BBD, although further study is needed to confirm the results of this pilot study and to assess relative contributions of sympathetic and parasympathetic function in producing pupillary abnormalities. This study has several limitations, including the small sample size, the absence of data on severity and duration of symptoms, and the absence of a control group of patients without any voiding symptoms. A simple tool for diagnosing BBD and for monitoring response to treatment could significantly improve the quality of treatment for one of the most common pediatric urologic complaints. Given the heterogeneity of symptoms under the BBD umbrella, pupillometric data could guide selection of treatment options, as well as assess adequacy of response to pharmacologic therapy.

Diazepam in a patient with chronic schizophrenia complicated by agoraphobia.

A case of chronic schizophrenia complicated by agoraphobia expressed as an exacerbation of negative symptoms is presented. The patient responded to diazepam combined with behavior therapy. The need to recognize treatable factors contributing to negative symptoms of schizophrenia is discussed.

Carbamazepine in bipolar-depressed disorder complicated by tricyclic antidepressant switching: case report.

The use of carbamazepine in a depressed bipolar patient with a history of manic switching with tricyclic antidepressants is described. Carbamazepine effectively treated the depression and mania was avoided.

Distribution of Bratton-Marshall-positive material in mice following intravenous injections of nitrosoureas.

As determined by a colorimetric assay measuring parent compounds plus ether-extractable nitroso-containing metabolites, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) disappeared more rapidly than N-(2-chloroethyl)-N'cyclohexyl-N-nitrosourea (CCNU) and N-(2-chloroethyl)-N'-(4-transmethylcyclohexyl)-N-nitrosourea (MeCCNU) and their products from the tissues of mice injected IV. Assay of selected samples by high-pressure liquid chromatography revealed that the colorimetric assay for BCNU was specific in that the two assays gave equivalent results. Following IV-injections of CCNU and MeCCNU, however, levels of the parent compounds decreased much more rapidly than the total, color-producing material. Of seven tissues, the largest Cxt values for BCNU, as determined by the colorimetric assay, were noted for blood (442 microgram-min/ml) and large intestine (285 microgram-min/g). Liver (29 microgram-min/g) had the lowest Cxt value, reflecting rapid metabolism of the compound by this organ. Color-producing material related to CCNU disappeared from the solid tissues of mice in a manner generally parallel to that for blood. Of the Cxt values for this compound and its products, those for lung (1753 microgram-equivalents-min/g), kidney (1633 microgram-equivalents-min/g), and small intestine (1557 microgram-equivalents-min/g) were highest. Consistent with its slower rate of metabolism, MeCCNU and its color-producing metabolites remained in most tissues of mice for 12 h following injection. Except for brain (1434 microgram-equivalents-min/g), Cxt values for this nitrosourea and its metabolites in tissues were higher than those of blood (5485 microgram-equivalents-min/ml), with kidney (15,324 micrograms-equivalents-min/g), liver (12,921 microgram-equivalents-min/g), and large intestine (11,501 microgram-equivalents-min/g) being highest. For each nitrosourea, a fair correlation was observed between the Cxt values for tissues and the toxic and/or antitumor effects at those sites.

Relationship among depression scores, beta-endorphin, and angina pectoris during exercise in patients with coronary artery disease.

OBJECTIVES: To determine the relationship between depression scores and (1) anginal indices during exercise including time to onset of angina, duration of angina, and severity of angina and (2) beta-endorphin at rest and in response to exercise. DESIGN: Prospective clinical trial. SETTING: Tertiary-care university hospital. PATIENTS: Fifty-eight patients with documented coronary artery disease and exercise-induced ischemia. OUTCOME MEASURES: Anginal indices during exercise (time to onset of angina, duration of angina, severity of angina); hemodynamic measures (systolic blood pressure, heart rate, rate pressure product) at rest, at onset of angina and at peak exercise; and plasma beta-endorphin levels at rest and immediately after exercise. RESULTS: Twenty-two of 58 patients had typical angina and electrocardiographic change indicating myocardial ischemia during exercise. There was a positive correlation between depression scores and duration of angina and a negative correlation between depression scores and time to onset of angina. Among patients with angina during exercise, systolic blood pressure increased to a greater extent in patients with high depression scores. Patients with high depression scores had higher resting beta-endorphin levels. CONCLUSIONS: Patients with depressed mood had greater perception of anginal pain than nondepressed patients, which cannot be explained by differences in the severity of ischemia. Possible mechanisms include an alteration in beta-endorphin regulation or differences in baroreceptor stimulation.

Fluorometric assay of thioguanine.

A spectrophotofluorometric method is described for the assay of thioguanine. The assay involves the oxidation of thioguanine with potassium permanganate to a fluorescent product. The method is suitable for routine quality control in laboratory preparations of thioguanine.

Fluorometric assay for measuring biological half-life of coralyne sulfoacetate in dogs and monkeys.

A spectrophotofluorometric assay for coralyne sulfoacetate was developed. Caralyne was extracted from serum samples with n-butyl alcohol, and the drug concentrations were determined fluorometrically at 475 nm when the extract was excited at 325 nm. A biphasic serum decay curve for coralyne was observed for both dogs and monkeys. The biological half-lives for the two phases were 20 and 196 min in dogs and 15 and 142 min in monkeys.

Tarsal tunnel syndrome.

The diagnosis of tarsal tunnel syndrome can be difficult to make. Clinical findings may be varied and symptoms are commonly vague and diffuse. MR imaging, with its excellent soft tissue contrast and ability to demonstrate musculotendinous and neurovascular structures, clearly demonstrates the anatomy of the tarsal tunnel and its contents and the presence and extent of lesions causing this syndrome.

Red blood cell labeling with technetium-99m. Effect of radiopaque contrast agents.

Radiographic contrast agents have been reported in the literature to interfere significantly with red blood cell (RBC) labeling in vivo by Tc-99m. Moreover, in the presence of contrast agents, red cells have been known to undergo significant morphologic changes. These observations led to the current RBC labeling study in patients (N = 25) undergoing procedures with the administration of contrast media. Before and after contrast administration, blood samples were drawn from each patient into vacutainer tubes containing heparin and RBC labeling was performed using 1-ml aliquots of these samples following the Brookhaven National Laboratory protocol. The differences in average percentage labeling yield with and without contrast media were not significant. In vivo labeling in hypertensive rats with administration of contrast media up to 600 mg likewise consistently gave high labeling yields at all concentrations. Purported alterations in cell labeling attributed to contrast agents are not reflected in these studies, and other pathophysiologic factors need to be identified to substantiate the previous reports. In vitro study offers a potentially useful and simple method to delineate effects of various agents on cell labeling.

A guideline for the design of a four-wheeled walker.

More people use assistive technology devices to compensate for mobility impairments than for any other general type of impairment. Increasing numbers of people with mobility or balance problems use walkers with four wheels. Four-wheeled walkers are often outfitted with seats to make it possible to travel longer distances with intermediate resting periods. The dangers of sitting on a parked walker are well known. Many physiotherapists tell walker users to park the walker against a wall to prevent injury in case the user forgets to apply the brakes or the brakes fail. To design a safer walker that can be used for sitting, the demands placed on it must be measured. With these data, three modes of walker instability must be considered: first, the brakes may hold but the wheels may slide along the ground; second, the entire walker may tip over; and third, the brakes may fail to hold the wheels in place, and they may begin to roll. Mathematical models can be constructed to simulate how different walker designs will perform. By this process, design improvements can be made for existing walkers, and future walker designs can also be proposed.

Cell-based interventions for neurologic conditions: ethical challenges for early human trials.

BACKGROUND: Attempts to translate basic stem cell research into treatments for neurologic diseases and injury are well under way. With a clinical trial for one such treatment approved and in progress in the United States, and additional proposals under review, we must begin to address the ethical issues raised by such early forays into human clinical trials for cell-based interventions for neurologic conditions. METHODS: An interdisciplinary working group composed of experts in neuroscience, cell biology, bioethics, law, and transplantation, along with leading disease researchers, was convened twice over 2 years to identify and deliberate on the scientific and ethical issues raised by the transition from preclinical to clinical research of cell-based interventions for neurologic conditions. RESULTS: While the relevant ethical issues are in many respects standard challenges of human subjects research, they are heightened in complexity by the novelty of the science, the focus on the CNS, and the political climate in which the science is proceeding. CONCLUSIONS: Distinctive challenges confronting US scientists, administrators, institutional review boards, stem cell research oversight committees, and others who will need to make decisions about work involving stem cells and their derivatives and evaluate the ethics of early human trials include evaluating the risks, safety, and benefits of these trials, determining and evaluating cell line provenance, and determining inclusion criteria, informed consent, and the ethics of conducting early human trials in the public spotlight. Further study and deliberation by stakeholders is required to move toward professional and institutional policies and practices governing this research.

The epidural dorsomedian septum as a possible cause for unilateral anaesthesia in an infant.

Unilateral epidural anaesthesia occurring in an infant is reported. An epidurogram revealed the presence of a midline structure suggestive of the dorsomedian septum. Epidural anatomy is reviewed and implications for threading epidural catheters in infants are discussed.

Determination of quinidine in serum by spectrofluorometry, liquid chromatography and fluorescence scanning thin-layer chromatography.

Quinidine is determined in serum by direct and extraction spectrofluorometry, by reflectance fluorescence scanning thin-layer chromatography (TLC), and by high-performance liquid chromatography (HPLC). Least-squares analyses of patients' sera (n = 62) analyzed first by direct fluorometry (x) and then HPLC (y) gave a slope of 0.52, an y-intercept of -0.40, a standard error of estimate of 0.65, and a correlation coefficient of 0.83. Comparison of patients' sera (n = 59) determined by extraction fluorometry (x) and then HPLC (y) gave a slope of 0.998, an y-intercept of -0.175, a standard error of estimate of 0.30, and a correlation coefficient of 0.96. Comparison of patients' sera (n = 36) by HPLC (x) and then reflectance fluorescence scanning TLC (y) gave a slope of 0.837, an y-intercept of 0.152, and a correlation coefficient of 0.94. Methaqualone and oxazepam interfere with HPLC. Within-run precision is 1.6, 1.0, 5.2 and 3.0% by direct fluorometry, extraction fluorometry, TLC and HPLC while between-run precision is 5, 3.5, 9 and 6.0%, respectively.

The predictive value of indium-111 leukocyte scans in the diagnosis of infected total hip, knee, or resection arthroplasties.

To evaluate the usefulness of the indium-111 scan in detecting actually or potentially infected total hip, knee, and resection arthroplasties, 153 scans were performed on 143 patients who underwent reoperation for a loose or painful total joint arthroplasty or a resection arthroplasty between 1990 and 1996. Scans were interpreted as infected, not infected, or equivocal by an experienced nuclear medicine radiologist. Patients were considered to be infected if they met any 2 of the following criteria: i) positive intraoperative cultures, ii) final permanent histologic section indicating acute inflammation, and iii) intraoperative findings of gross purulence within the joint. Twenty-six patients (17%) met the infection criteria at the time of reoperation. Indium scans were found to have a 77% sensitivity, 86% specificity, 54% and 95% positive and negative predictive values, and 84% accuracy for the prediction of infection. Of 6 equivocal scans, none were infected. The results of this study suggest limited indications for the use of the indium-111 scan in the evaluation of painful hip, knee, or resection arthroplasties. A negative indium scan may be helpful in suggesting the absence of infection in cases in which the diagnosis is not otherwise evident.