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.

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.

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.

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.

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.

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.

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.

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.

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.

The Ranawat Award. Sulcus morphology of the distal femur.

Traditional images of the distal femur place the intercondylar groove midway between the condyles. The location of the sulcus of the intercondylar groove in a large Sudanese skeletal population was verified using a custom stereotactic device. The results of this study show that the femoral sulcus is lateral to the midplane between the 2 femoral condyles. This study also shows that the configuration of the sulcus is linear and is oriented between the traditional anatomic and mechanical axes of the femur.

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.

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.

Human aminoacylase-1. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer.

Human aminoacylase-1 (N-acyl-L-amino-acid amidohydrolase, EC 3.5.1.14; ACY1) is a homodimeric zinc-binding enzyme that catalyzes the hydrolysis of N alpha-acylated amino acids. ACY1 has been assigned to chromosome 3p21.1, a region reduced to homozygosity in small cell lung cancer (SCLC), and has been reported to exhibit reduced or absent expression in SCLC cell lines and tumors. Two human cDNA libraries and one human genomic DNA library were screened with a previously isolated partial ACY1 cDNA to isolate a full-length transcript. Sequence analysis of clones from each of these libraries resulted in an ACY1 cDNA of 1438 base pairs with an open reading frame of 1224-base pairs coding for a putative protein of 408 amino acids with a predicted molecular mass of 45,882 Da. Sequence analysis revealed no homologies to previously reported cDNA or protein sequences and establishes ACY1 as the first member of a new family of zinc-binding enzymes to be so characterized. The subcellular location of ACY1 has been established as cytosolic by flow cytometry. Southern and northern analyses of ACY1 in SCLC cell lines failed to demonstrate any gross abnormalities of the ACY1 structural gene or instances of absent or aberrantly sized mRNA, respectively.

Segmental stenosis of the renal artery: pattern recognition of tardus and parvus abnormalities with duplex sonography.

Segmental renal artery branches within the renal sinus were prospectively evaluated with color Doppler imaging and pulsed-Doppler spectral analysis in 56 patients before angiography. Waveforms were evaluated for the tardus and parvus abnormalities of prolonged acceleration time, diminished acceleration index, and loss of the normal early systolic compliance peak/reflective-wave complex (ESP). Findings obtained with these parameters were compared with the subsequent findings on angiograms to ascertain their efficacy in detection of hemodynamically significant (greater than or equal to 60%) renal arterial stenosis (RAS), which was present in 32 kidneys in 26 patients. Simple pattern-recognition analysis of ESP proved to be the best of the three parameters. Loss of ESP enabled identification of RAS with 95% sensitivity, 97% specificity, a 92% positive predictive value, a 98% negative predictive value, a 96% overall accuracy. On the basis of the high technical success rate, high sensitivity and specificity, and short examination time, waveform analysis for detection of tardus-parvus abnormalities, especially loss of ESP, of the segmental artery is recommended as an alternative to direct examination of the main renal arteries for evaluation of RAS.

MR imaging of symptomatic peripheral vascular malformations.

We performed a retrospective study of symptomatic peripheral vascular malformations to determine if MR imaging can be used to distinguish slow-flow venous malformations from high-flow arteriovenous malformations and arteriovenous fistulas. Twenty-seven MR examinations in 25 patients with malformations outside the CNS were reviewed. Sixteen venous malformations, nine arteriovenous malformations, and two arteriovenous fistulas were included. In all cases, the MR findings were correlated with the results of angiography. The distinction between slow-flow venous malformations and high-flow arteriovenous malformations and arteriovenous fistulas was made primarily on T2-weighted MR images, which showed high signal intensity in venous malformations and flow voids in high-flow lesions. In addition to the previously described MR features of venous malformations (serpentine pattern with septations, associated muscle atrophy, and typical T1 and T2 signal intensities), several new MR features were apparent. Venous malformations had a propensity for multifocal involvement (37%), orientation along the long axis of extremities or affected muscles (78%), and adherence to neurovascular distributions (64%). Prominent subcutaneous fat was commonly seen adjacent to the malformation. MR images of arteriovenous malformations and arteriovenous fistulas also commonly showed muscle atrophy and subcutaneous fatty prominence. Our results show that slow-flow venous malformations can be distinguished from high-flow arteriovenous malformations and fistulas on the basis of spin-echo MR signal characteristics. The associated imaging characteristics help in the differential diagnosis in problematic cases.

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.

Nonpalpable breast lesions: stereotactic automated large-core biopsies.

One hundred two patients with mammographically suspicious, nonpalpable lesions underwent stereotactic breast biopsy with a biopsy gun and an automated 14-gauge cutting needle. After biopsy, a localization wire was placed and surgical biopsy performed. There was agreement of the histologic results from the gun biopsy and the surgical biopsy specimens in 98 cases (96%), including 22 of 23 carcinomas (96%) (kappa = 0.936). The gun biopsy yielded findings that led to the correct diagnosis in two cases involving lesions that were missed at surgical biopsy; two lesions found at surgery were missed at gun biopsy. The results of this study suggest that the use of 14-gauge needles improves agreement between surgical and needle core biopsy findings and that stereotactic biopsy with an automated needle and gun can be an acceptable alternative to surgical biopsy in women with mammographically suspicious breast lesions.