The effects of frozen tissue storage conditions on the integrity of RNA and protein.

Unfixed tissue specimens most frequently are stored for long term research uses at either -80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at -80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA integrity and mRNA integrity. Protein stability was analyzed by surface-enhanced or matrix-assisted laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS, MALDI-TOF-MS) and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RNA yield and total RNA integrity showed significantly better results for -80° C storage compared to VPLN storage; the transcripts that were preferentially degraded during VPLN storage were these involved in antigen presentation and processing. No consistent differences were found in the SELDI-TOF-MS, MALDI-TOF-MS or nLC-ESI-MS/MS analyses of specimens stored for more than 8 years at -80° C compared to those stored in VPLN. Long term storage of human research tissues at -80° C provides at least the same quality of RNA and protein as storage in VPLN.

Identification of gyrB and rpoB gene mutations and differentially expressed proteins between a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain and its virulent parent strain.

A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23 kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection.

Shear waves in viscoelastic wormlike micellar fluids over a broad concentration range.

Low frequency (61 Hz) shear wave speeds have been measured in viscoelastic wormlike micellar (WM) fluids for a concentration range of 20/12-500/300 mM CTAB/NaSAL where CTAB is the surfactant and NaSAL is the salt and the concentration ratio was fixed at 0.6 for all experiments. The birefringent property of the WM fluids was exploited to visually track the the shear pulse using crossed optical polarizing filters and high speed video. Several scalings of shear wave speed as a function of concentration were discovered: c(s) ~ √C for 20-200 mM and c(s) ~ C for higher concentrations, but with a break in the slope at 400 mM CTAB. Over this full concentration range, the shear wave speed varied from 0.08-0.7 m/s. The shear wave speed was also found to be sensitive to the time between fluid synthesis and measurement indicating a long equilibrium time. Further, comparison with elastic and loss moduli obtained from rheology data show that shear wave propagation is dominated by the elastic modulus for this frequency range. Also briefly discussed are potential applications of this fluid in elastography.

Shear waves in viscoelastic wormlike micellar fluids.

Low frequency (61 Hz) shear wave speeds have been measured in viscoelastic wormlike micellar (WM) fluids for a concentration range of 20/12-160/96 mM CTAB/NaSAL. The strain induced birefringence of the WM fluids was exploited to optically track the shear pulse using crossed polarizing filters and high speed video. It was found that shear speed increases roughly linearly with concentration at a rate of 3.5 mm s(-1) mM(-1) CTAB. Further, comparison with elastic and loss moduli obtained from rheology data show that shear wave propagation is dominated by the elastic modulus for this frequency range.

Emissions inventory of PM2.5 trace elements across the United States.

This paper presents the first National Emissions Inventory (NEI) of fine particulate matter (PM2.5) that includes the full suite of PM2.5 trace elements (atomic number > 10) measured at ambient monitoring sites across the U.S. PM2.5 emissions in the NEI were organized and aggregated into a set of 84 source categories for which chemical speciation profiles are available (e.g., Unpaved Road Dust Agricultural Soil, Wildfires). Emission estimates for ten metals classified as Hazardous Air Pollutants (HAP) were refined using data from a recent HAP NEI. All emissions were spatially gridded, and U.S. emissions maps for dozens of trace elements (e.g., Fe, Ti) are presented for the first time. Nationally, the trace elements emitted in the highest quantities are silicon (3.8 x 10(5) ton/yr), aluminum (1.4 x 10(5) ton/yr), and calcium (1.3 x 10(5) ton/yr). Our chemical characterization of the PM2.5 inventory shows that most of the previously unspeciated emissions are comprised of crustal elements, potassium, sodium, chlorine, and metal-bound oxygen. This work also reveals that the largest PM2.5 sources lacking specific speciation data are off-road diesel-powered mobile equipment, road construction dust, marine vessels, gasoline-powered boats, and railroad locomotives.

Air emission inventories in North America: a critical assessment.

Although emission inventories are the foundation of air quality management and have supported substantial improvements in North American air quality, they have a number of shortcomings that can potentially lead to ineffective air quality management strategies. Major reductions in the largest emissions sources have made accurate inventories of previously minor sources much more important to the understanding and improvement of local air quality. Changes in manufacturing processes, industry types, vehicle technologies, and metropolitan infrastructure are occurring at an increasingly rapid pace, emphasizing the importance of inventories that reflect current conditions. New technologies for measuring source emissions and ambient pollutant concentrations, both at the point of emissions and from remote platforms, are providing novel approaches to collecting data for inventory developers. Advances in information technologies are allowing data to be shared more quickly, more easily, and processed and compared in novel ways that can speed the development of emission inventories. Approaches to improving quantitative measures of inventory uncertainty allow air quality management decisions to take into account the uncertainties associated with emissions estimates, providing more accurate projections of how well alternative strategies may work. This paper discusses applications of these technologies and techniques to improve the accuracy, timeliness, and completeness of emission inventories across North America and outlines a series of eight recommendations aimed at inventory developers and air quality management decision-makers to improve emission inventories and enable them to support effective air quality management decisions for the foreseeable future.

A 1000+ channel bionic communication system.

The wireless electronic nervous system interface known as the functional electrical stimulation-battery powered bion system is being developed at the Alfred Mann Foundation. It contains a real-time propagated wave micro-powered multichannel communication system. This system is designed to send bi-directional messages between an external master controller unit (MCU), and each one of a group of injectable stimulator-sensor battery powered bion implants (BPB). The system is capable of communicating in each direction about 90 times per second using a structure of 850 time slots within a repeating 11 millisecond time window. The system's total Time Division Multiple Access (TDMA) communication capability is about 77,000 two-way communications per second on a single 5 MHz wide radio channel. Each time slot can be used by one BPB, or shared alternately by two or more BPBs. Each bidirectional communication consists of a 15 data bit message sent from the MCU sequentially to each BPB and 10 data bit message sent sequentially from each BPB to the MCU. Redundancy bits are included to provide error detection and correction. This communication system is designed to draw only a few microamps from the 3.6 volt, 3.0 mAHr lithium ion (LiIon) battery contained in each BPB, and the majority of the communications circuitry is contained within a 1.4x5 mm integrated circuit.

Innovative methods for emission inventory development and evaluation: workshop summary.

Emission inventories are an essential tool for evaluating, managing, and regulating air pollution. Refinements and innovations in instruments that measure air pollutants, models that calculate emissions, and techniques for data management and uncertainty assessment are needed to enhance emission inventories. This workshop provided recommendations for improving emission factors, improving emission models, and reducing inventory uncertainty. Communication that increases cooperation between developers and users of inventories is essential. Emission inventories that incorporate these improvements will meet the challenges of the future.

A novel diagnostic test for prostate cancer emerges from the determination of alpha-methylacyl-coenzyme a racemase in prostatic secretions.

PURPOSE: With the recent discovery that alpha-methlyacly-coenzyme A racemase (AMACR) is over expressed in a majority of prostate cancer (CaP) specimens we developed a novel polymerase chain reaction (PCR) based approach that would predict the presence of CaP from prostatic secretions. MATERIALS AND METHODS: A total of 21 patients were enlisted in this study, including 10 with CaP, 2 with high grade PIN and 9 cancer-free individuals (7 healthy men and 2 with benign hyperplasia). Total cellular RNA was extracted from prostatic secretions obtained from post-massage urine specimens. Levels of AMCAR transcripts and prostate specific antigen (PSA) transcripts in these samples were determined by quantitative reverse transcriptase-PCR analyses. Relative AMACR value scores (RAVSs) were calculated by normalizing the AMACR transcript level to that of PSA for each sample and multiplying by 100. An experimentally defined diagnostic cutoff RAVS value was determined in the cancer-free control group. RESULTS: Neither AMACR nor PSA mRNA levels were predictive of CaP when used alone. However, using RAVS values and imposing a diagnostic cutoff value of 2 SDs above the mean RAVS in the cancer-free control group all 9 (100%) cancer-free individuals, including those with benign prostatic hyperplasia, were below the cutoff and 7 of 10 (70%) with CaP had RAVS above the cutoff. Furthermore, 2 of the 3 false-negative cases showed clinically insignificant disease. The 2 patients with high grade PIN were above the cutoff in this test. CONCLUSIONS: In this study the quantification of AMACR transcripts normalized to PSA transcripts in prostatic secretions was shown to be predictive of CaP. Therefore, our novel approach using quantitative reverse transcriptase-PCR to detect the AMACR-to-PSA transcript ratio shows promise as a noninvasive screening test for CaP. Furthermore, early results demonstrate a trend toward excluding patients with clinically insignificant disease that may not yet require aggressive treatment due to a low cancer burden.

Monitoring the serological proteome: the latest modality in prostate cancer detection.

PURPOSE: Various strategies have recently emerged to improve the diagnostic prediction of prostate cancer (CaP). One such strategy includes the mass profiling of serum protein fractions selectively adsorbed onto chemically modified probes. In the current study we further validated this approach, while offering a more versatile, less expensive and yet equally predictive alternative to existing technologies. MATERIALS AND METHODS: A solid core lipophilic C-18 resin was used to extract and enrich the low molecular weight protein fraction from patient serum for further analysis by mass spectrometry. Mass spectra generated from a 48 patient training set were data mined using multivariate analysis to identify diagnostically significant protein peaks. These peaks were then used to test a blinded study set comprising 168 patients with common statistical algorithms and commercially available software packages. RESULTS: A total of 36 peaks generated from the training set were used to test the combined set of 168 serum samples obtained from 98 healthy individuals and 70 patients with CaP. We report a sensitivity of 94.1% and a specificity of 99.0% with 1 false-positive, 4 false-negative and 5 nondiagnosed cases. CONCLUSIONS: Our results further indicate that mass profiling of serological proteins provides a means for the accurate detection of CaP. In addition, our approach was found to be superior to chip based protocols, generating rich, sharp, highly reproducible spectra attainable in a high throughput manner and at minimal cost. This technique is also scaleable for subsequent protein characterization using multidimensional protein identification technologies. Finally, analyses of mass spectra with commercially available statistical applications was found to be highly effective in generating highly discriminatory m/z values for CaP diagnosis.

Battery powered BION FES network.

The Alfred Mann Foundation is completing development of a coordinated network of BION microstimulator/sensor (hereinafter implant) that has broad stimulating, sensing and communication capabilities. The network consists of a master control unit (MCU) in communication with a group of BION implants. Each implant is powered by a custom lithium-ion rechargeable 10 mW-hr battery. The charging, discharging, safety, stimulating, sensing, and communication circuits are designed to be highly efficient to minimize energy use and maximize battery life and time between charges. The stimulator can be programmed to deliver pulses in any value in the following range: 5 microA to 20 mA in 3.3% constant current steps, 7 micros to 2000 micros in 7 micros pulse width steps, and 1 to 4000 Hz in frequency. The preamp voltage sensor covers the range 10 microV to 1.0 V with bandpass filtering and several forms of data analysis. The implant also contains sensors that can read out pressure, temperature, DC magnetic field, and distance (via a low frequency magnetic field) up to 20 cm between any two BION implants. The MCU contains a microprocessor, user interface, two-way communication system, and a rechargeable battery. The MCU can command and interrogate in excess of 800 BlON implants every 10 ms, i.e., 100 times a second.

Radiation dosimetry using three-dimensional optical random access memories.

The ability to determine particle type and energy plays an important role in the dosimetry of heavy charged particles (HCP) and neutrons. A new approach to radiation dosimetry is presented, which is shown to be capable of particle type and energy discrimination. This method is based on utilising radiation induced changes in the digital information stored on three-dimensional optical random access memories (3D ORAM). 3D ORAM is a small cube (a few mm3) composed of poly(methyl methacrylate) doped with a photochromic dye. and it was originally proposed as a memory device in high speed parallel computers. A Nd:YAG laser system is used to write and read binary information (bits) on the ORAM, which functions as a charged particle detector. Both the read and the write processes use two laser beams that simultaneously strike the material to cause a colour change at their intersection (similar to the darkening of light-sensitive sunglasses when exposed to sunlight.) The laser produces colour changes in the ORAM, which then reverts to the original colour ('bit-flips') at sites where energy is deposited from interaction with incident HCP or neutron-recoil protons. The feasibility of this approach was demonstrated both theoretically and experimentally. Calculations based on track structure theory (TST) predict that when HCP interact with the ORAM material, the local energy deposition is capable of inducing measurable 'bit-flips'. These predictions were recently confirmed experimentally using two types of ORAM systems, one based on spirobenzopyran and the other on anthracene, as the photochromic dyes.

Laser-induced fluorescence studies of polycyclic aromatic hydrocarbons (PAH) vapors at high temperatures.

In this work, we present the fluorescence spectra of anthracene and pyrene vapors at different elevated temperatures (from 150 to 650 degrees C) excited with the 337 nm line of a nitrogen laser. We describe the high temperature effects on the resulting spectral properties including spectral intensity, spectral bandwidth and spectral shift. We found that the PAH fluorescence spectral bandwidths become very broad as the temperature increases. The broadening is mainly due to thermal vibrational sequence congestion. We also have found that the fluorescence intensity of pyrene vapor increases with increasing temperature, which results from the increase of the pyrene vapor absorption cross section at 337 nm.

Measurement of cellular adhesion under static conditions.

This unit describes procedures for measuring the adhesion of lymphoid cells to immobilized ligands or adherent cell monolayers. The basic protocol, which is optimized for human T cells, can be easily modified to study almost any cell type in suspension if the corresponding adhesive ligand is available. Alternate protocols describe modifications to the basic protocol that allow analysis of cell adhesion in the presence of stimulators of lymphocyte activation or monoclonal antibodies (MAbs) that react with adhesion molecules or their ligands, or analysis of cell adhesion to adherent cell monolayers. A support protocol is provided for determining the optimal concentration of adhesion ligands and choosing the appropriate type of microtiter plate for static adhesion assays.

Effects of phytoestrogens and synthetic combinatorial libraries on aromatase, estrogen biosynthesis, and metabolism.

Approximately 60% of breast cancer patients have hormone-dependent breast cancer containing estrogen receptors and requiring estrogen for tumor growth. The extent of estrogen biosynthesis and metabolism in the breast cancer tissue microenvironment influences breast-tumor development and growth, and endogenous and exogenous agents may alter the levels of hormonally active estrogens and their metabolites. Isoflavonoid phytoestrogens such as genistein exhibit numerous biochemical activities; however, their effects on estrogen biosynthesis and metabolism in breast cancer cells have not been fully examined. MCF-7 cells (hormone-dependent) and MBA-MB-231 cells (hormone-independent) were treated with genistein (100 nM) for five days and then incubated with radiolabeled estradiol (100 nM, 2.5 microCi) for 0 to 48 h. Media were extracted with ethyl acetate, and the organic residues analyzed by reverse-phase HPLC with a radioactivity flow detector. The major metabolite formed in all cases is estrone, although differences were observed between the cell lines and the various drug treatments. The formation of estrone in untreated MCF-7 cells (approximately 9.3% of radioactivity at 24 h) is relatively limited, in contrast to untreated MDA-MB-231 cells (approximately 32.0% of radioactivity at 24 h). Treatment of MCF-7 cells with 100 nM genistein increased the conversion of estradiol to estrone up to 19.5% in 24 h. The effect of genistein on estrone formation in MDA-MB-231 cells resulted in 37.7% of the radioactivity being estrone. Thus, genistein treatment of breast cancer cells resulted in increased 17-betahydroxysteroid dehydrogenase activity and elevated formation of estrone. Increased levels of oxidative 17-betahydroxysteroid dehydrogenase activity (Type II) were confirmed by Western blots. Therefore, exposure of breast cancer cells to genistein results in elevated conversion of estradiol to estrogenically weaker or inactive metabolites. The regulation of breast-tissue aromatase by exogenous agents such as drugs and environmental agents is being investigated. The benzopyranone-ring system is a molecular scaffold of considerable interest, and this scaffold is found in flavonoid natural products that have weak aromatase inhibitory activity. Medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aro- matase inhibitors. Several compounds in the initial libraries have demonstrated moderate aromatase inhibitory activity in screening assays.

Kramers-Kronig relations applied to finite bandwidth data from suspensions of encapsulated microbubbles

In this work, the Kramers-Kronig (K-K) relations are applied to experimental data of resonant nature by limiting the interval of integration to the measurement spectrum. The data are from suspensions of encapsulated microbubbles (Albunex) and have the characteristics of an ultrasonic notch filter. The goal is to test the consistency of this dispersion and attenuation data with the Kramers-Kronig relations in a strict manner, without any parameters from outside the experimental bandwidth entering in to the calculations. In the course of reaching the goal, the artifacts associated with the truncation of the integrals are identified and it is shown how their impacts on the results can be minimized. The problem is first approached analytically by performing the Kramers-Kronig calculations over a restricted spectral band on a specific Hilbert transform pair (Lorentzian curves). The resulting closed-form solutions illustrate the type of artifacts that can occur due to truncation and also show that accurate results can be achieved. Next, both twice-subtracted and lower-order Kramers-Kronig relations are applied directly to the attenuation and dispersion data from the encapsulated microbubbles. Only parameters from within the experimental attenuation coefficient and phase velocity data sets are used. The twice-subtracted K-K relations produced accurate estimates for both the attenuation coefficient and dispersion across all 12 data sets. Lower-order Kramers-Kronig relations also produced good results over the finite spectrum for most of the data. In 2 of the 12 cases, the twice-subtracted relations tracked the data markedly better than the lower-order predictions. These calculations demonstrate that truncation artifacts do not overwhelm the causal link between the phase velocity and the attenuation coefficient for finite bandwidth calculations. This work provides experimental evidence supporting the validity of the subtracted forms of the acoustic K-K relations between the phase velocity and attenuation coefficient.

On the applicability of Kramers-Kronig relations for ultrasonic attenuation obeying a frequency power law

In the recent literature concern has been raised regarding the validity of Kramers-Kronig relations for media with ultrasonic attenuation obeying a frequency power law. It is demonstrated, however, that the Kramers-Kronig dispersion relations for application to these types of media are available. The developed dispersion relations are compared with measurements on several liquids, and agreement is found to better than 1 m/s over the experimentally available bandwidth. A discussion regarding the validity of these dispersion relations, in particular how the dispersion relations relate to the so-called Paley-Wiener conditions, forms the conclusion.

Antibody-based biosensor for breast cancer with ultrasonic regeneration.

We describe a novel method and instrumental setup for regenerating antibodies immobilized on a fiberoptic probe of an immunosensor using ultrasonic irradiation with broadband imaging transducers. The instrumental setup and irradiation conditions for antibody regeneration using ultrasound are described. The results of the measurements with antibody against breast cancer antigen illustrate the effectiveness and potential of the regenerable immunosensor. A 65% removal of the antigens bound to the Mab immobilized on the fiber surface is attained after ultrasound regeneration.

Cesarean delivery for suspected fetal distress among preterm parturients.

OBJECTIVE: Among preterm parturients (< 37 weeks) who underwent cesarean delivery for suspected fetal distress, to determine the factors associated with decision-incision time (DIT) of < or = 30 minutes and to assess if umbilical arterial pH < 7.10 is more common with DIT < or = 30 or > 30 minutes. STUDY DESIGN: The peripartum course of all patients who had cesareans for suspected fetal distress over three years was reviewed. The inclusion criteria were reliable gestational age < 37 weeks and a single indication for cesarean delivery, suspected fetal distress. Twenty antepartum and intrapartum factors were used in a univariate analysis. RESULTS: The mean DIT among the 84 parturients was 30.5 +/- 21.2 minutes, and 63% of patients had surgery started within 30 minutes. The incidence of pH < 7.10 was 20%. Multivariate analysis indicated that the two factors significantly associated with prolonged time to surgery were tachycardia with decreased variability (odds ratio [OR] 5.9, 95% confidence interval [CI] 1.6-21.6) and use of spinal anesthesia (OR 6.2, 95% CI 1.1-35.0). Though none of the 20 variables had significant univariate associations with neonatal acidosis at alpha = .05, those with P < .20 were considered in multiple logistic regression analysis. None of the 20 factors were associated with pH < 7.10, including DIT of > or = 30 minutes (OR 0.26, 95% CI 0.06-1.03). CONCLUSION: DIT is likely to be > 30 minutes if cesarean delivery is due to decreased fetal heart variability or if spinal anesthesia is utilized; neonatal acidosis, however, is not significantly associated with a prolonged interval.

Three-dimensional optical random access memory materials for use as radiation dosimeters.

This article describes the development of the first three-dimensional optical random access memory (3D-ORAM) material and readout system for monitoring energetic neutrons. Two different photochromic dyes, 5'-chloro-6-nitro-1',3',3'-trimethylspiro-[2H-1-benzopyran-2,2'-in doline] (spirobenzopyran) and anthracene, have been investigated for use in these 3-D ORAM dosimeter materials. These dyes were immobilized in a poly(methyl methacrylate) support, and the resulting dosimeter materials were irradiated with neutrons from a Cf-252 source. Fluorescence measurements from the dosimeter show a dramatic decrease in the overall fluorescence intensity of the 3D-ORAM dosimeter exposed to the Cf-252, relative to a nonirradiated dosimeter. In addition, a two-photon excitation readout system has been developed for determining characteristics of the radiation that are necessary for estimating dose.