In Silico Discovery and Validation of Neuropeptide-Y-Binding Peptides for Sensors.

Wearable sensors for human health, performance, and state monitoring, which have a linear response to the binding of biomarkers found in sweat, saliva, or urine, are of current interest for many applications. A critical part of any device is a biological recognition element (BRE) that is able to bind a biomarker at the surface of a sensor with a high affinity and selectivity to produce a measurable signal response. In this study, we discover and compare 12-mer peptides that bind to neuropeptide Y (NPY), a stress and human health biomarker, using independent and complimentary experimental and computational approaches. The affinities of the NPY-binding peptides discovered by both methods are equivalent and below the micromolar level, which makes them suitable for application in sensors. The in silico design protocol for peptide-based BREs is low cost, highly efficient, and simple, suggesting its utility for discovering peptide binders to a variety of biomarker targets.

Skeletal and cardiac muscle defects in a murine model of Emery-Dreifuss muscular dystrophy.

Previous histological findings, physiological data, and behavioral observations on the A-type lamin knockout mouse (Lmna(-/-)) suggest that important aspects of this model resemble the human Emery-Dreifuss muscular dystrophy (EDMD) phenotype. The main goal of our experiments was to study skeletal and cardiac muscle function in this murine model to obtain the semiquantitative data needed for more detailed comparisons with human EDMD defects. Measurements of the mechanical properties of preparations from two different skeletal muscle groups, the soleus and the diaphragm, were made in vitro. In addition, records of the electrocardiogram, and measurements of heart rate variability were obtained; and phasic contractions (unloaded shortening) of enzymatically isolated ventricular myocytes were monitored. Soleus muscles from Lmna(-/-) mice produced less force and work than control preparations. In contrast, force and work production in strips of diaphragm were not changed significantly. Lead II electrocardiograms from conscious, restrained Lmna(-/-) mice revealed slightly decreased heart rates, with significant prolongations of PQ, QRS, and 'QT' intervals compared with those from control recordings. These ECG changes resemble some aspects of the ECG records from humans with EDMD; however, the cardiac phenotype in this Lmna(-/-) mouse model appears to be less well-defined/developed. Ventricular myocytes isolated from Lmna(-/-) mice exhibited impaired contractile responses, particularly when superfused with the beta-adrenergic agonist, isoproterenol (1 microM). This deficit was more pronounced in myocytes isolated from the left ventricle(s) than in myocytes from the right ventricle(s). In summary, tissues from the Lmna(-/-) mouse exhibit a number of skeletal and cardiac muscle deficiencies, some of which are similar to those which have been reported in studies of human EDMD.

1-(substituted-benzyl)imidazole-2(3H)-thione inhibitors of dopamine beta-hydroxylase.

Molecular shape and quantitative structure-activity relationship (QSAR) analyses of 52 1-(substituted-benzyl)-imidazole-2(3H)-thione inhibitors of dopamine beta-hydroxylase were carried out. QSARs were developed for sets of 45 and sets of 47 analogues. Molecular shape, as represented by common overlap steric volume and the composite charge density on carbons 3, 4, and 5 of the substituted-benzyl ring are the major inhibition-potency descriptors. Five of the 52 compounds were eliminated prior to analyses on the basis of difficulties in characterizing shape and charge state. Two compounds were outliers. The active conformation deduced in the analyses is a low-energy conformer for both active and inactive inhibitors. This suggests that the intrinsic shape of the molecule due to the selection of X is more important than torsion-angle selection for the bonds between the two rings. The QSARs found in this study have only general similarities to one put forth by Kruse et al. using linear free energy descriptors.

A generalized formalism of three-dimensional quantitative structure-property relationship analysis for flexible molecules using tensor representation.

A general formalism, based upon tensor representation of multidimensional data blocks, is presented to express relationships between dependent properties and independent molecular feature measures. The solutions to these data set problems are three-dimensional quantitative structure-property relationships, 3D-QSPRs. The molecular features are partitioned into the intrinsic molecular shape tensor, the molecular field tensor, a nonshape/field feature tensor, and an experimental feature tensor. The intrinsic molecular shape tensor contains information on the shape of a molecule within the contact surface while the molecular field tensor contains information outside of the contact surface. Molecular features not directly related to molecular shape are put into the nonshape/field tensor. Experimental measures not being used as dependent variables can be considered as independent molecular features in the experimental feature tensor. The 3D-QSPR is realized by constructing the transformation tensor which optimizes the statistical significance between the dependent and independent variables. Use of partial least squares (PLS) regression permits the unfolding of the composite feature tensor and the identification of the optimum transformation tensor. It is pointed out that a variety of fragment, whole-molecule, two-dimensional, and/or three-dimensional features can be placed into a nonshape/field tensor.

Construction of a molecular shape analysis-three-dimensional quantitative structure-analysis relationship for an analog series of pyridobenzodiazepinone inhibitors of muscarinic 2 and 3 receptors.

A generalized three-dimensional (3D) quantitative structure-property relationship (QSPR) formalism, based upon molecular shape analysis (MSA), has been applied to an analog series of pyridobenzodiazepinone inhibitors of muscarinic 2 (M2) and 3 (M3) receptors. The fundamental goal of this application is to establish MSA-3D-QSARs (P = A = inhibition activity) that are based upon identifying the active conformations of these flexible analogs. The repetitive use of partial least squares (PLS) analysis permits the construction of the MSA-3D-QSARs. In addition to molecular shape, the identification of the properties of a lipophilic binding site and specific nonallowed steric receptor sites govern the MSA-3D-QSARs. The M2 and M3 QSARs suggest receptor subtype specificity might be realized by targeting upon a specific nonallowed steric receptor site. One conformation, common to both M2 and M3 receptors, emerges as dominant in the optimum MSA-3D-QSARs. However, other similar conformations are also found to yield meaningful MSA-3D-QSARs.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

The precision of a modern method for body compartment measurements using multiple isotopes.

Using readily available beta and gamma detectors, combined measurements of exchangeable sodium, exchangeable potassium, total body water and extracellular fluid volume using the isotopes 24Na, 43K, 3H and 35S were made in ten normal subjects. These measurements were repeated 1-6 weeks later so that an index of precision for each isotope study could be calculated. For such measurements the precision was 55 mmol for exchangeable sodium, 149 mmol for exchangeable potassium, 1.26 litres for total body water and 1.11 litres for extracellular fluid volume. The precision of our method is the best so far reported using readily available apparatus. These results may encourage further studies on different pathological conditions in man to be undertaken in peripheral laboratories.

Body space measurements in the hyponatraemia of carcinoma of the bronchus: evidence for the chronic 'sick cell' syndrome?

Body space measurements using simultaneous multiple isotope dilution techniques were made in both hyponatraemic and normonatraemic patients with carcinoma of the bronchus and wasting, and compared with those in a group of normal volunteers. Both groups of patients showed osmolal loss from cells. The significance of these findings in relation to the development of hyponatraemia is discussed.

The retinol-binding protein system: a potential paradigm for steroid-binding globulins?

Retinol (vitamin A) is an example of a small molecule that is essential for higher organisms; its utilisation has been involved in the evolution of a number of proteins. In mammalian species, retinol is obtained from the diet and controls the release of its binding protein from hepatocytes into the blood stream. Subsequent influx into cells under normal situations usually involves a specific membrane-bound receptor for retinol-binding protein, which facilitates the uptake of retinol alone or bound to its carrier. This specific receptor has not yet been identified, but a receptor for a related lipocalin has been cloned. It represents a relatively new family, and there are a number of related genes in various eukaryotic genomes, suggesting that the system is very widespread in multicellular organisms. Its significance has been highlighted recently by the suggestion that retinol-binding protein, through its receptor, may play a major role in type 2 diabetes, perhaps the greatest scourge of modern society. This system may provide a new paradigm in mammalian biology, another example of which may exist in the processes responsible for steroid handling. This review outlines the characteristics of retinol utilisation in mammalian species, focusing primarily on the uptake system.

Stimulation of residual insulin secretion by glibenclamide in insulin dependent diabetics.

Residual insulin secretion, reflected by the presence of C-peptide in serum and urine, has been demonstrated in 5 of 10 insulin-requiring diabetics of less than 10 years' duration tested. The C-peptide response, in the C-peptide secretors, showed a significant increase in both serum and urine after 4 weeks' treatment with 15 mg glibenclamide daily in addition to their usual insulin regime although no beneficial effects in metabolic control were detected. It is suggested that glibenclamide might be a useful adjunct to insulin therapy in insulin-requiring diabetics who still secrete C-peptide.

Electrophoretically mediated microanalysis of beta-galactosidase on microchips.

Electrophoretically mediated microanalysis (EMMA) is a method of accomplishing chemical analyses, typically in an open-tubular capillary, due to the difference in the electrophoretic mobility between the particular reagents. This work reports on combining this technique onto microfabricated systems. Two methods of this technique were applied, constant potential and zero potential EMMA onto chips. A dosage response curve was run using this constant potential mode that resulted in a linear response over three orders of substrate concentration magnitude. The chemical system used here is beta-galactosidase (beta-Gal) as the enzyme and fluorescein mono-beta-D-galactopyranoside (FMG) as the substrate. The zero potential mode was used to amplify product turnover using various incubation times. Using this technique and a 10 min incubation, approximately 40000 enzyme molecules could be detected. The zero potential mode is also used in conjunction with an internal standard to show how one can quantitate using this method. The power and ease of utility of this technique is described.

Ureteral jets in normal second- and third-trimester pregnancy.

PURPOSE: We wished to assess whether urodynamic changes accompanying normal pregnancy altered the pattern of ureteral jets, complicating detection of pathologic obstruction. METHODS: Ureteral jets were observed with color Doppler sonography for 5 minutes in 26 women in the second or third trimester of pregnancy and in 6 non-pregnant controls (3 men and 3 women). RESULTS: A mean of 5.5 jets/minute were detected in the pregnant subjects, and the mean difference in frequency of jets between the right and left sides was 42%. Corresponding results for controls were 7.6 jets/minute and 11%, respectively. The 2 groups were significantly different with respect to jet symmetry (p < 0.02). Unilateral absence of jets was noted in 4 pregnant women but in no controls. CONCLUSIONS: Because of variation in ureteral jet bilaterality and symmetry during the later stages of pregnancy, caution is recommended in the use of the technique to diagnose obstructive urolithiasis in this population.

The radiation dose to man following the intravenous injection of radiosulphate (Na2(35)SO4).

An estimation of the radiation dose to mans' testes, the critical organ, for radiosulphate (Na2(35)SO4) has been made by a combination of studies on rats and human volunteers. The radiation dose to rats testes and the rate of disappearance of radiosulphate from the blood and urine of humans have been increased. The calculated radiation dose of 7.7 muGy/MBq to mans' testes is lower than previously reported.

Hyperlipidaemia diagnosed at lumbar puncture.

A patient presenting with subarachnoid haemorrhage and high lipid concentrations in the cerebrospinal fluid (taken at lumbar puncture), who has later shown to have type V hyperlipidaemia is described. This case, so far as can be ascertained by the authors, is the first report of hyperlipidaemia being diagnosed from CSF examination.

Improved diabetic control in insulin-dependent diabetics treated with insulin and glibenclamide.

A randomized double blind trial of insulin and glibenclamide treatment vs insulin and placebo was carried out in 20 insulin-dependent diabetics. Nine patients were C-peptide secretors and all increased their C-peptide output during a 50 g OGTT at the end of the insulin and glibenclamide treatment period by a mean of 47%. At the same time their mean daily blood glucose fell from 8.4 +/- 1.7 to 7.4 +/- 1.5 mmol/l and their HbA1 from 8.1 +/- 0.5 to 7.5 +/- 0.9% (mean +/- SD). There was no change in any of the measurements of diabetic control in C-peptide non-secretors and no evidence for any extra-pancreatic effects of glibenclamide in this group of patients. Combined insulin and glibenclamide treatment may produce a useful improvement of diabetic control in insulin-dependent diabetics who still secrete some endogenous insulin, although further studies are required.

Electrophoretically mediated microanalysis of leucine aminopeptidase using two-photon excited fluorescence detection on a microchip.

Two-photon excited fluorescence detection was performed on a microfabricated electrophoresis chip. A calibration curve of the fluorescent tag beta-naphthylamine was performed, resulting in a sensitivity of 2.5 x 10(9) counts M(-1) corresponding to a detection limit of 60 nM. Additionally, leucine aminopeptidase was assayed on the chip using electrophoretically mediated microanalysis. The differential electroosmotic mobilities of the enzyme and substrate, L-leucine beta-naphthylamide, allowed for efficient mixing in an open channel, resulting in the detection of a 30 nM enzyme solution under constant potential. A zero potential incubation for 1 min yielded a calculated detection limit of 4 nM enzyme.

A picoliter-volume mixer for microfluidic analytical systems.

Mixing confluent liquid streams is an important, but difficult operation in microfluidic systems. This paper reports the construction and characterization of a 100-pL mixer for liquids transported by electroosmotic flow. Mixing was achieved in a microfabricated device with multiple intersecting channels of varying lengths and a bimodal width distribution. All channels running parallel to the direction of flow were 5 microm in width whereas larger 27-microm-width channels ran back and forth through the parallel channel network at a 45 degrees angle. The channel network composing the mixer was approximately 10 microm deep. It was observed that little mixing of the confluent solvent streams occurred in the 100-microm-wide, 300-microm-long mixer inlet channel where mixing would be achieved almost exclusively by diffusion. In contrast, after passage through the channel network in the approximately 200-microm-length static mixer bed, mixing was complete as determined by confocal microscopy and CCD detection. Theoretical simulations were also performed in an attempt to describe the extent of mixing in microfabricated systems.

Evaluation of palpable breast masses with color Doppler sonography and gray scale imaging.

Excisional biopsy is the standard method of distinguishing benign from malignant masses of the breast. However, alternative, less invasive methods of diagnosis are needed to reduce the number of unnecessary biopsies, allay anxiety of the patient, and control costs. In this study, we evaluated breast masses in a series of patients using color Doppler sonography and gray scale ultrasonographic features. In all cases, the pathologic diagnosis of the breast mass was subsequently established by excisional biopsy. The accuracy of gray scale sonography exceeded that of color Doppler sonography at a significance level of P < 0.005.

Stereotaxic core breast biopsy: value in providing tissue for flow cytometric analysis.

OBJECTIVE: Stereotaxic core biopsy provides intact samples of breast tissue for accurate histologic analysis. We conducted a study to determine if prognostic data could also be successfully derived from such core samples and how the data correlate with surgical biopsy. MATERIALS AND METHODS: Both core and surgical breast biopsies from 135 patients were processed under a uniform flow cytometry protocol. Samples were coded and then randomly processed at an outside flow cytometer and interpreted by an independent pathologist; the code was broken and patients' results correlated only after all samples were completely analyzed. RESULTS: Core breast biopsy provides intact tissue that can be successfully processed by a flow cytometer, even after being embedded in paraffin for initial histologic analysis. Larger cores (14 gauge) had fewer insufficient samples, as recorded on ploidy histograms. Although ploidy may reflect the underlying aggressiveness of a lesion and assist in evaluating breast cancer, surgical-pathologic correlation with stereotaxic biopsy indicated, as has been confirmed in other studies, considerable overlap of different ploidy types between benign and malignant conditions. There was no correlation between mammographic presentation and ploidy or S-phase fractions. CONCLUSION: Stereotaxic large-core biopsy can enable accurate histologic diagnosis of breast disease and furnish sufficient tissue for flow cytometric measurements of ploidy and S-phase fractions, even at an interval following paraffinization. Such prognostic information aids in planning of adjuvant therapy, allows flexibility should surgery fail to provide enough tissue for DNA study, and helps radiologists further market stereotaxic biopsy to clinicians.

Assessment of the metabolic effects of dietary carbohydrate and fibre by measuring urinary excretion of C-peptide.

An assessment of the metabolic effect of dietary carbohydrate on daily insulin secretion, reflected by the 24-h urine output of C-peptide, has been made. Urinary C-peptide excretion increased when the carbohydrate intake of 6 normal subjects was increased from 200 to 400 g. A high-fibre diet rich in beans and lentils caused a significant fall in urine C-peptide and a lowering of blood glucose values in both normal and diabetic subjects. This confirms the insulin-sparing effect of leguminous foods. An increase of dietary fibre to 50 g mainly as cereal fibre did not significantly alter urine C-peptide excretion in normal subjects. Urinary C-peptide estimations could be useful for assessing the effect of different diets on daily insulin secretion.