High-Q Magnetic Levitation and Control of Superconducting Microspheres at Millikelvin Temperatures.

We report the levitation of a superconducting lead-tin sphere with 100 µm diameter (corresponding to a mass of 5.6 µg) in a static magnetic trap formed by two coils in an anti-Helmholtz configuration, with adjustable resonance frequencies up to 240 Hz. The center-of-mass motion of the sphere is monitored magnetically using a dc superconducting quantum interference device as well as optically and exhibits quality factors of up to 2.6×10^{7}. We also demonstrate 3D magnetic feedback control of the motion of the sphere. The setup is housed in a dilution refrigerator operating at 15 mK. By implementing a cryogenic vibration isolation system, we can attenuate environmental vibrations at 200 Hz by approximately 7 orders of magnitude. The combination of low temperature, large mass, and high quality factor provides a promising platform for testing quantum physics in previously unexplored regimes with high mass and long coherence times.

Pharmacotherapy for depressed pregnant women: overcoming obstacles to gathering essential data.

Approximately 3% of pregnant women take antidepressant medications. Information on the impact of antidepressants on short- and long-term maternal and offspring outcomes is highly desirable but neglected. The position that the dearth of treatment information is of greater concern than the risks to pregnant subjects involved in medical research is gaining support. Mandating the collection of reproductive outcome data in exposed childbearing women is an overdue step toward societal responsibility to our most vulnerable members.

Improving women's health through modernization of our bioinformatics infrastructure.

Our nationwide bioinformatics infrastructure used to detect important sex differences associated with medical product use is antiquated. The Food and Drug Administration (FDA) has embarked on an ambitious bioinformatics modernization effort that will improve our ability to assess the safety and effectiveness of new medical products. This, in turn, will improve our ability to detect important sex differences.

Females in clinical studies: where are we going?

Historically females of all ages have been underrepresented in clinical research. Reasons for this exclusion are multifactorial and may possibly have their origins in regulation that forbid the participation of females of childbearing potential in the earliest phase clinical studies that support drug approval. Decades of female underrepresentation in clinical studies has resulted in inequality in the understanding, diagnosis, and treatment of disease between the sexes. Adequate numbers of both sexes is one approach which is likely to present overwhelming financial constraints. Advances in study design, statistical methodologies, and the promise of evolving technologies will lead to new tools that can foster a better understanding of the biology that governs sex and gender differences.

Advancing women's health via FDA Critical Path Initiative.

Studying sex and gender differences is critical to understanding diseases that affect women solely, disproportionately or differently from men. Although inclusion of both sexes is essential in clinical research, advanced technology and analysis methods offer tools to define complex biological and physicochemical differences and improve prevention, diagnosis and treatments for diseases in women and men. This paper identifies the potential for biomarker development, pharmacogenetics and bioinformatics in research under the FDA Critical Path Initiative.:

Nebulization of liposomal rh-Cu/Zn-SOD with a novel vibrating membrane nebulizer.

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.

Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation.

The overproduction of biochemical mediators, and activation of leukocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. The formation of short-lived, highly reactive metabolites, such as oxygen free radicals, increases with increasing tissue ischemia, and causes further cell damage. Human recombinant Cu/Zn-superoxide dismutase (rh-Cu/Zn-SOD), an enzyme which captures these radicals, may have a beneficial effect on the postburn inflammation processes. In this study, the influence of rh-Cu/Zn-SOD application to thermally injured tissue of rabbit backskin was examined. Three different delivery strategies were compared, pure or liposomally encapsulated enzyme, or intralesionally injected rh-Cu/Zn-SOD. For control, one animal group was treated with plain gel and another group was kept untreated. At 24 h following trauma a statistically significant difference in lesion sizes between the enzyme treated and control groups was observed. After 72 h tissue swelling had diminished significantly more in the rh-Cu/Zn-SOD treated groups as compared to the control animals. The best results were achieved by spreading liposomes encapsulating the enzyme onto the wounds. Our results suggest that local treatment of burn wounds with enzymatic radical scavengers such as rh-Cu/Zn-SOD has a beneficial effect on the extent of the postburn damage.

A newly created splice donor site in exon 25 of the MyBP-C gene is responsible for inherited hypertrophic cardiomyopathy with incomplete disease penetrance.

BACKGROUND: Hypertrophic cardiomyopathy is a myocardial disorder resulting from inherited sarcomeric dysfunction. We report a mutation in the myosin-binding protein-C (MyBP-C) gene, its clinical consequences in a large family, and myocardial tissue findings that may provide insight into the mechanism of disease. METHODS AND RESULTS: History and clinical status (examination, ECG, and echocardiography) were assessed in 49 members of a multigeneration family. Linkage analysis implicated the MyBP-C gene on chromosome 11. Myocardial mRNA, genomic MyBP-C DNA, and the myocardial proteins of patients and healthy relatives were analyzed. A single guanine nucleotide insertion in exon 25 of the MyBP-C gene resulted in the loss of 40 bases in abnormally processed mRNA. A 30-kDa truncation at the C-terminus of the protein was predicted, but a polypeptide of the expected size ( approximately 95 kDa) was not detected by immunoblot testing. The disease phenotype in this family was characterized in detail: only 10 of 27 gene carriers fulfilled diagnostic criteria. Five carriers showed borderline hypertrophic cardiomyopathy, and 12 carriers were asymptomatic, with normal ECG and echocardiograms. The age of onset in symptomatic patients was late (29 to 68 years). In 2 patients, outflow obstruction required surgery. Two family members experienced premature sudden cardiac death, but survival at 50 years was 95%. CONCLUSIONS: Penetrance of this mutation was incomplete and age-dependent. The large number of asymptomatic carriers and the good prognosis support the interpretation of benign disease.

Delayed-type hypersensitivity skin reactions caused by subcutaneous unfractionated and low-molecular-weight heparins: tolerance of a new recombinant hirudin.

BACKGROUND: Eczema-like infiltrated plaques at subcutaneous heparin injection sites are well-documented side effects of these anticoagulants. However, surgical interventions may be problematic if heparin is urgently needed in these patients. OBJECTIVE: The aims of this study were to perform extensive allergy skin testing in 24 patients, including a pregnant woman in whom subcutaneous infiltrated plaques developed after subcutaneous heparin injections, and to find safe therapeutic alternatives for this group of patients. METHODS: Patch, intradermal, and subcutaneous tests were performed with a panel of unfractionated heparins (UFHs), low-molecular-weight heparins (LMWHs), and heparinoids. Since 1997, we have also been performing allergy studies in 8 patients with lepirudin, a new recombinant heparinoid; tolerance of lepirudin was investigated by means of subcutaneous and intravenous injections. The allergy investigations in the pregnant woman were limited to patch tests with heparins and intradermal and subcutaneous tests with pentosanpolysulfate, which are not contraindicated during pregnancy. RESULTS: In our study population 19 of 23 patients were sensitized to all the UFHs and LMWHs tested when intracutaneous and subcutaneous test results were read at up to 96 hours. LMWH was found to be a possible substitute in 4 patients. Five patients were also sensitized to the heparinoid pentosanpolysulfate. Sensitization to the heparinoid danaparoid was observed in 12 of the 13 patients who were tested with this substance. The administration of an intravenous bolus containing a therapeutic dose of lepirudin after negative subcutaneous provocation was tolerated without any side effects in all 8 patients. The pregnant woman was sensitized to LMWH but tolerated subcutaneous pentosanpolysulfate without any side effects. CONCLUSION: Extensive allergy skin testing should be performed to find safe alternatives. With few exceptions, all patients react to both UFHs and LMWHs, as well as to danaparoid. The subcutaneous provocation test is the most reliable diagnostic measure. Intravenous lepirudin, and in some cases subcutaneous pentosanpolysulfate, appears to be a safe alternative in patients with eczema-like infiltrated plaques at subcutaneous heparin-injection sites.

Determination of liposome size distribution by flow cytometry.

BACKGROUND: An essential parameter that describes the quality of liposome suspensions is the mean size, respectively the size distribution. Currently several analytical methods including laser light scattering techniques (LLST) are being employed. METHODS: Here we present an alternative technique using flow cytometry (FCM) to characterize uni- and polydisperse suspensions. As model liposomes preparations containing dipalmitoylphosphatidylcholine (DPPC) were used. A constant number of particles (1,500/s) in the fluid stream and a representative number of 10,000 particles of each sample was measured. Fluorescence-labeled latex beads were measured identically, and their side scatter signals were calibrated and correlated to the results obtained with liposome vesicles. RESULTS: Evaluation of the measurement and validation of the FCM results in comparison to LLST confirm the reliability of results obtained with our method. Latex beads in the range of 100-1000 nm were used for calibration to classify liposomes. Although measurement characteristics and calculation in both methods are basically different, very good agreement of the results was achieved. CONCLUSIONS: Demonstration of stability, reproducibility, and reliability of results make the employment of this method acceptable for an adequate routine analysis technique.

Genomic structure and fine mapping of the two human filamin gene paralogues FLNB and FLNC and comparative analysis of the filamin gene family.

The genomic structure of the filamin gene paralogues FLNB and FLNC was determined and related to FLNA. FLNB consists of 45 exons and 44 introns and spans approximately 80 kb of genomic DNA. FLNC is divided into 48 exons and 47 introns and covers approximately 29.5 kb of genomic DNA. A previously unknown intron was found in FLNA. The comparison of all three filamin gene paralogues revealed a highly conserved exon-intron structure with significant differences in the exons 32 of all paralogues encoding the hinge I region, as well as the insertion of a novel exon 40A in FLNC only. Gene organization does not correlate with the domain structures of the respective proteins. To improve candidate gene cloning approaches, FLNB was precisely mapped at 3p14 in an interval of 0.81 cM between WI3771 and WI6691 and FLNC at 7q32 in an interval of 2.07 cM between D7S530 and D7S649.

S100-Beta, melanoma-inhibiting activity, and lactate dehydrogenase discriminate progressive from nonprogressive American Joint Committee on Cancer stage IV melanoma.

PURPOSE: Monitoring advanced malignant melanoma, serum levels of S100-beta (S100beta) and melanoma-inhibiting activity (MIA) were assessed for the ability to discriminate progressive from nonprogressive disease. S100beta and MIA were supposed to be superior to conventional variables, such as lactate dehydrogenase (LDH) level. PATIENTS AND METHODS: Seventy-one patients with stage IV malignant melanoma according to the criteria of the American Joint Committee on Cancer (AJCC) were included in the study. Results of restaging examinations were used as an independent reference standard for diagnosing progressive disease, and S100beta, MIA, LDH level, and erythrocyte sedimentation rate (ESR) were determined in venous blood just before restaging. Sensitivities and specificities of the parameters were calculated by logistic regression analysis. Discrimination ability was assessed by Somers' D(xy) rank correlation and the area under the receiver-operating characteristic curve (ROC-AUC). RESULTS: All tested serum parameters were significantly elevated in patients with progressive disease. The highest sensitivities according to the established thresholds were found for S100beta and MIA (91% and 88%, respectively). LDH had the highest specificity (92%). ESR was dropped from the analysis because of low specificity. In calculating Somers' D(xy) and ROC-AUC values, S100beta, MIA, and LDH showed high discrimination ability. By multiple logistic regression, LDH was identified to be the only statistically significant marker for progressive disease. S100beta and MIA did not provide additional significant information because of their high correlation with LDH with respect to clinical outcome. CONCLUSION: Elevated serum levels of S100beta, MIA, and LDH indicate current disease progression in AJCC stage IV melanoma. LDH was the most relevant overall parameter.

Effects of Plasmodium berghei infection on cytochromes P-450 2E1 and 3A2.

Metabolism and disposition of most drugs used to treat malaria are substantially altered in malaria infection. Few data are available that specify effects of malaria infection on drug metabolism pathways in humans or animal model systems. In this report, studies were undertaken to determine the effect of Plasmodium berghei infection on cytochrome P-450 (CYP450) 2E1 and 3A2-mediated metabolism and enzyme expression in rat liver microsomes. Malaria infection (MAL) resulted in significant decreases in total cytochrome P-450 content (56%, P < 0.05) and NADPH cytochrome P-450 reductase activity (32%, P < 0.05) as compared to control (CON) rats. Chlorzoxazone 4-hydroxylase activity (CYP2E1-mediated) showed no significant difference between CON and MAL microsomes while testosterone 6-beta-hydroxylase activity (CYP3A2-mediated) was reduced by 41% (P < 0.05) in MAL. Enzyme kinetic studies and immunoblot analysis indicate that the loss of activity for CYP3A2 in malaria infection is due to significantly decreased CYP3A2 protein expression. The altered expression of CYP450s in malaria infection should be taken into account when treating patients with malaria in order to minimize drug-drug interactions or toxicity.

Analysis of mitogenic activity of proteins after separation by gel electrophoresis.

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions.