Quasi-static acoustic tweezing thromboelastometry.

Essentials Blood coagulation measurement during contact with an artificial surface leads to unreliable data. Acoustic tweezing thromboelastometry is a novel non-contact method for coagulation monitoring. This method detects differences in the blood coagulation state within 10 min. Coagulation data were obtained using a much smaller sample volume (4 µL) than currently used. SUMMARY: Background Thromboelastography is widely used as a tool to assess the coagulation status of critical care patients. It allows observation of changes in material properties of whole blood, beginning with early stages of clot formation and ending with clot lysis. However, the contact activation of the coagulation cascade at surfaces of thromboelastographic systems leads to inherent variability and unreliability in predicting bleeding or thrombosis risks. Objectives To develop acoustic tweezing thromboelastometry as a non-contact method for perioperative assessment of blood coagulation. Methods Acoustic tweezing is used to levitate microliter drops of biopolymer and human blood samples. By quasi-statically changing the acoustic pressure we control the sample drop location and deformation. Sample size, deformation and location are determined by digital imaging at each pressure. Results Simple Newtonian liquid solutions maintain a constant, reversible location vs. deformation curve. In contrast, the location/deformation curves for gelatin, alginate, whole blood and blood plasma uniquely change as the samples solidify. Increasing elasticity causes the sample to deform less, leading to steeper stress/strain curves. By extracting a linear regime slope, we show that whole blood or blood plasma exhibits a unique slope profile as it begins to clot. By exposing blood samples to pro- or antithrombotic agents, the slope profile changes, allowing detection of hyper- or hypocoagulable states. Conclusions We demonstrate that quasi-static acoustic tweezing can yield information about clotting onset, maturation and strength. The advantages of small sample size, non-contact and rapid measurement make this technique desirable for real-time monitoring of blood coagulation.

Modeling cavitation nucleation from laser-illuminated nanoparticles subjected to acoustic stress.

In an earlier work by Farny et al. [ARLO 6, 138-143 (2005).] it was demonstrated that the acoustic cavitation threshold in a tissue mimicking gel phantom can be lowered from 4.5 to ∼1 MPa by "seeding" the optically transparent phantom with light absorptive gold nanoparticles and irradiating these absorbers with nanosecond pulses of laser light at intensities less than 10 mJ/cm(2). As a follow-up study, a three-stage numerical model was developed to account for prenucleation heating, the nucleation and formation of the vapor cavity, and the resulting vapor bubble dynamics. Through examination of the radius-time evolution of the cavity, the combined thresholds for laser radiant exposure and acoustic peak pressure required to induce inertial cavitation are deduced. It is found that the threshold pressure decreases when laser exposure increases; but the rate depends on exposure levels and the size of the particle. Investigations of the roles of particle size and laser pulse length are performed and optimum choices for these parameters determined in order to obtain inertial cavitation at the lowest possible acoustic pressure and laser intensity.

Measurements of bubble-enhanced heating from focused, MHz-frequency ultrasound in a tissue-mimicking material.

Time-resolved measurements of the temperature field in an agar-based tissue-mimicking phantom insonated with a large aperture 1-MHz focused acoustic transducer are reported. The acoustic pressure amplitude and insonation duration were varied. Above a critical threshold acoustic pressure, a large increase in the temperature rise during insonation was observed. Evidence for the hypothesis that cavitation bubble activity in the focal zone is the cause of enhanced heating is presented and discussed. Mechanisms for bubble-assisted heating are presented and modeled, and quantitative estimates for the thermal power generated by viscous dissipation and bubble acoustic radiation are given.

Signal sequence and alanine-rich region of streptococcal protein antigen A of Streptococcus sobrinus can direct localization of alkaline phosphatase to the periplasm of Escherichia coli.

Streptococcal protein antigen A (SpaA) of Streptococcus sobrinus is expressed on the surface of cells and extracellularly. TnphoA which lacks signals for transcription and membrane transport of Escherichia coli alkaline phosphatase was used to analyze the sequences necessary for transport of a SpaA/PhoA fusion protein across the cytoplasmic membrane to the periplasm of E. coli cells. Of 15 alkaline phosphatase-producing isolates analyzed, all were found to localize more than 85% of the SpaA/PhoA hybrid protein to the periplasm of E. coli cells. From DNA sequence analysis, all were found to have TnphoA inserted into an identical site. The insertion site of TnphoA was downstream from the coding sequence that generates four tandemly repeated alanine-rich sequences of 82 amino acid residues. These results suggest that in addition to the signal sequence, mature protein sequences containing alanine-rich repeat sequences may play a role in the export of the SpaA protein across a bacterial membrane.

Experimental observations of bubble response and light intensity near the threshold for single bubble sonoluminescence in an air-water system.

Single bubble sonoluminescence in an air-water system has been shown to occur along a unique surface in the acoustic pressure-ambient radius-gas concentration parameter space where the bubble is stable both in shape and in (average) size. In this paper, we show how the bubble deviates from the expected path (traced by the shape-instability threshold as a function of pressure) in order to reach the observed stability. We also present measurements of the expansion ratio (R(max)/R(0)) for bubbles near the threshold for light emission. The results suggest that maximal bubble radial response is an insufficient criterion for the onset of light emission, and we present data for the dependence of the emitted light on several parameters.

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing.

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.

An efficient cost-effective protocol for automated fluorescent-DNA sequencing.

A simple and cost-effective method is described to sequence double-stranded DNA (< 100 ng) using a very small amount of fluorescent sequencing mix (one-tenth of manufacturer-recommended amount). The modification has significant advantages over conventional protocols and can be used to sequence, using a single sequencing kit, at least ten times the number of samples. This modified method also removes the post-reaction cleaning protocol and can be used for direct loading of samples on gels after completion of sequencing reactions. The accuracy of DNA sequencing data from analysis of 700 to 750 nucleotides is greater than 98.5%.

Characterization of a P1-deficient strain of Streptococcus mutans that expresses the SpaA protein of Streptococcus sobrinus.

The Streptococcus sobrinus SpaA protein and the Streptococcus mutans P1 protein share 66% sequence homology at the amino acid level. To determine if the SpaA protein can be expressed in S. mutans and functionally replace the P1 protein, the spaA gene of S. sobrinus 6715 was isolated from plasmid pX1303 and inserted into the Escherichia coli-Streptococcus shuttle vector pVA838. The resulting plasmid pX1600 was transformed into the P1-deficient strain S. mutans 834 that has defects in saliva-mediated aggregation and in the ability to adhere to saliva-coated hydroxyapatite surfaces. Western blot (immunoblot) analysis of cellular protein fractions of S. mutans 834 (pX1600) detected in mutanolysin-solubilized cell walls a major protein of 210 kDa with an electrophoretic mobility similar to that of S. sobrinus SpaA protein and a minor 210-kDa protein and a major 64-kDa protein in the extracellular protein fraction. Analysis of virulence traits showed that expression of SpaA protein by S. mutans 834(pX1600) cells had restored the ability of the S. mutans 834 cells to aggregate in the presence of saliva or salivary agglutinin but not to adhere to saliva-coated hydroxyapatite. This cell aggregation was inhibited specifically by antisera to S. sobrinus SpaA protein. These results indicate that SpaA plays a role in the virulence of S. sobrinus by specifically interacting with fluid-phase salivary agglutinin to mediate cell aggregation.

Localization on the cell surface of streptococcal protein antigen A.

The cellular localization of the major surface protein SpaA of Streptococcus sobrinus 6715 was examined by immunoelectron microscopy with rabbit polyclonal antibodies directed against purified SpaA protein. Immunoelectron microscopic analysis of thin sections of S. sobrinus cells revealed that the SpaA protein is associated with the fibrillar fuzzy coat of S. sobrinus cells and appears to be distributed over the entire surface of S. sobrinus cells.

MR imaging of the shoulder: appearance of the supraspinatus tendon in asymptomatic volunteers.

MR imaging has been shown to be accurate in the diagnosis of rotator cuff disruption and tear. Uncertainty remains about the significance of increased signal intensity in the critical zone of the supraspinatus tendon without visible disruption of tendon fibers and about the significance of other secondary findings commonly encountered with rotator cuff abnormalities, such as musculotendinous retraction or obliteration and fluid in the subacromial space. We evaluated proton density-weighted and T2-weighted coronal images (obtained on a 1.5-T superconductive MR imager) of 55 shoulders in 32 asymptomatic volunteers for signal intensity in the supraspinatus tendon, location of the musculotendinous junction, fluid in the subacromial-subdeltoid space, and appearance of the fat plane. In 89% of shoulders, the supraspinatus tendon showed focal, linear, or diffuse increased signal intensity with or without loss of the low-signal-intensity tendon margin on proton density-weighted images. None of these findings were confirmed on T2-weighted images. The musculotendinous junction was always located within an area 15 degrees medial to 30 degrees lateral to the highest point (12 o'clock) on the humeral head convexity. A peribursal fat plane was poorly defined or absent in 49%, and fluid in the subacromial-subdeltoid space was found in 20%. Increased signal intensity in the supraspinatus tendon on proton density-weighted images without a corresponding increase on T2-weighted images, the presence of small amounts of fluid in the subacromial space, and the lack of preservation of the subdeltoid fat plane are common findings in asymptomatic shoulders and by themselves are poor predictors of rotator cuff disease.

Molecular cloning and characterization of the spaB gene of Streptococcus sobrinus.

A gene of Streptococcus sobrinus 6715 (serotype g) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.

Magnetic resonance imaging of the postoperative lumbar spine.

Magnetic resonance (MR) imaging is a powerful diagnostic tool for patients with the failed back surgery syndrome. The multiplanar imaging capability, superior soft-tissue contrast resolution, and excellent tissue characterization are its major advantages. It displays the changes caused by surgical intervention as well as associated postoperative findings, many of which cause the failed back surgery syndrome. Most recently, gadolinium-DTPA-enhanced MR imaging of the spine, combined with noncontrast MR imaging, has shown heretofore unparallelled sensitivity and accuracy.

Magnetic resonance imaging of the shoulder: rationale and current applications.

Because it can demonstrate a wide range of tissue contrast with excellent resolution, magnetic resonance (MR) imaging has revolutionized imaging in many areas of the musculoskeletal system and has generated excitement among those interested in the painful shoulder. Shoulder impingement syndrome and glenohumeral instability constitute the two major categories of shoulder derangements. Correct diagnosis requires the use of appropriate pulse sequences and imaging planes, proper patient positioning, and a satisfactory surface coil. In addition the imager must have a thorough understanding of shoulder anatomy and pathology. We present a summary of the current status of MR imaging of the shoulder including technical, anatomic, and pathologic considerations and a review of the pertinent literature.

Mie scattering used to determine spherical bubble oscillations.

Linearly polarized laser light is scattered from an oscillating, acoustically levitated bubble, and the scattered intensity is measured with a suitable photodetector. The output photodetector current is converted into a voltage and digitized. For spherical bubbles, the scattered intensity I(rel)(R,theta,t) as a function of radius R and angle theta is calculated theoretically by solving the boundary value problem (Mie theory) for the water-bubble interface. The inverse transfer function R(I) is obtained by integrating over the photodetector solid angle centered at some constant theta. Using R(I) as a look-up table, the radius vs time [R(t)] response is calculated from the measured intensity vs time [I(exp)(R,t)].

Glenoid labrum: preliminary work with use of radial-sequence MR imaging.

The authors describe a magnetic resonance imaging method for examination of the glenoid labrum of the shoulder joint that utilizes a radial fast-imaging sequence. Seven shoulders were examined: a total of five in three healthy asymptomatic volunteers, one in a symptomatic patient not suspected of having a lesion of the glenoid labrum, and one in a patient with recurrent shoulder dislocation and surgical proof of an extensive tear of the labrum. The preliminary results suggest that this technique may advantageously demonstrate pathologic changes in the glenoid labrum and may contribute to the evaluation of the unstable and painful shoulder.

CT and MR of lateral disc herniation: typical appearance and pitfalls of interpretation.

Lateral disc herniation is a relatively uncommon manifestation of intervertebral disc disease, defined as herniation of disc material lateral to or into the vertebral foramen. Failure to recognize this lesion preoperatively may lead to unsuccessful surgical exploration. We describe here the radiologic findings in seven patients with lateral disc herniation. Three were evaluated with magnetic resonance imaging alone, three with computed tomography alone, and one with both modalities. Example of three entities which may mimic lateral disc herniation (pseudodisc protrusion of scoliosis, paravertebral masses, and volume averaging artifacts) are illustrated.

Hypertrophy of C-1 anterior arch: useful sign to distinguish os odontoideum from acute dens fracture.

Dens fractures are often difficult to detect radiographically because of overlapping structures. The lateral radiographs were examined of all patients at the authors' institution who demonstrated an abnormal odontoid process or an abnormal anterior arch of C-1. Six patients were found: Four had os odontoideum, one had rheumatoid arthritis, and one had a congenital defect in the posterior arch of C-1. All had hypertrophy of the C-1 anterior arch on the lateral view. The width of the anterior arch of C-1 and the cortical thickness of the anterior arch in these six patients were measured and compared with the findings in a control group of 20 patients; the measurements from the six patients were found to be significantly greater. Hypertrophy of the anterior arch of C-1 is a useful sign of a chronic pathologic condition at the atlantoaxial articulation, and in the setting of acute trauma it may be an important clue that prevents unnecessary invasive treatment for a mistaken diagnosis of fractured dens.

Molecular and immunochemical characterization of recombinant Escherichia coli containing the spaA gene region of Streptococcus sobrinus.

We identified and characterized a recombinant Escherichia coli containing the entire gene for surface protein antigen A (spaA) of Streptococcus sobrinus 6715. The recombinant E. coli was isolated from a cosmid gene bank of size-fractionated S. sobrinus DNA fragments, and recombinants expressing the SpaA protein were detected immunologically. Subcloning experiments showed that the DNA sequences encoding the SpaA protein could be isolated on two contiguous EcoRI fragments, 3.7 and 3.3 kilobases (kb) in size, both contained on a 16.2-kb BglII fragment. Southern blot hybridization experiments using the EcoRI fragments to probe genomic DNAs from various serotypes of the mutans group of streptococci revealed DNA sequence homology not only to S. sobrinus 6715 (serotype g) chromosomal DNA but also to S. sobrinus serotype d DNA. Weak hybridization signals to Streptococcus mutans serotypes c, e, and f and to Streptococcus cricetus serotype a were observed with the 3.3-kb EcoRI fragment. These results suggest that the coding sequence for the spaA gene is probably conserved in S. sobrinus strains. Plasmid-encoded polypeptides made in E. coli minicells revealed that transcription of the spaA gene was initiated on the 3.7-kb EcoRI fragment and that its product size was about 210 kilodaltons. The cloned SpaA protein was purified from the periplasmic protein of E. coli, and monospecific antiserum against the cloned product was prepared in rabbits. The data obtained from various physiochemical and immunological procedures allowed us to conclude that the coding sequence for the entire spaA gene of S. sobrinus 6715 had been successfully cloned in E. coli and that faithful expression of the cloned product could be obtained.

Heart contains receptors for dihydrotestosterone but not testosterone: possible role in the sex differential in coronary heart disease.

The sex differential in coronary heart disease is well documented but poorly understood. Previous studies have demonstrated receptors for dihydrotestosterone (DHT) in the myocardium and smooth muscle cells of arteries from a number of species. In this autoradiographic study, we further investigated and characterized the in vivo uptake and retention of the androgen binding in the male baboon. Adult castrated male baboons were injected with 1 micrograms/kg bw 3H-testosterone; 1 hr after the injection, the animals were rapidly exsanguinated while under anesthesia. The heart and arterial system were removed and processed for autoradiography. As a negative control, one animal received both 3H-testosterone and 100-fold unlabeled testosterone. For positive controls, the pituitary gland, prostate, seminal vesicles, and other tissues were also removed and processed for autoradiography. In contrast to our previous finding with 3H-DHT, no nuclear uptake and retention of 3H-steroid was found in any of the cells in either the heart or the arterial system. In the positive control tissues, pituitary gland, prostate, seminal vesicles, and others, a very distinct nuclear uptake and retention of 3H-steroid was observed, which was completely inhibited by the simultaneous injection of 100-fold unlabeled testosterone. In the binding study, Scatchard analysis of the cytosol prepared from a 17-year-old female baboon demonstrated levels of androgen receptor (as determined by the use of radiolabeled R1881) comparable to that found in young adults.(ABSTRACT TRUNCATED AT 250 WORDS)