Quantitative SPECT/CT Imaging of (177)Lu with In Vivo Validation in Patients Undergoing Peptide Receptor Radionuclide Therapy.

PURPOSE: The purpose of this study is to extend an established SPECT/CT quantitation protocol to (177)Lu and validate it in vivo using urine samples, thus providing a basis for 3D dosimetry of (177)Lu radiotherapy and improvement over current planar methods which improperly account for anatomical variations, attenuation, and overlapping organs. PROCEDURES: In our quantitation protocol, counts in images reconstructed using an ordered subset-expectation maximization algorithm are converted to kilobecquerels per milliliter using a calibration factor derived from a phantom experiment. While varying reconstruction parameters, we tracked the ratio of image to true activity concentration (recovery coefficient, RC) in hot spheres and a noise measure in a homogeneous region. The optimal parameter set was selected as the point where recovery in the largest three spheres (16, 8, and 4 ml) stagnated, while the noise continued to increase. Urine samples were collected following 12 SPECT/CT acquisitions of patients undergoing [(177)Lu]DOTATATE therapy, and activity concentrations were measured in a well counter. Data was reconstructed using parameters chosen in the phantom experiment, and estimated activity concentration from the images was compared to the urine values to derive RCs. RESULTS: In phantom data, our chosen parameter set yielded RCs in 16, 8, and 4 ml spheres of 80.0, 74.1, and 64.5 %, respectively. For patients, the mean bladder RC was 96.1 ± 13.2% (range, 80.6-122.4 %), with a 95 % confidence interval between 88.6 and 103.6 %. The mean error of SPECT/CT concentrations was 10.1 ± 8.3% (range, -19.4-22.4 %). CONCLUSIONS: Our results show that quantitative (177)Lu SPECT/CT in vivo is feasible but could benefit from improved reconstruction methods. Quantifying bladder activity is analogous to determining the amount of activity in the kidneys, an important task in dosimetry, and our results provide a useful benchmark for future efforts.

Two-beam coupling between filament-forming beams in air.

We experimentally demonstrate two-beam coupling between nearly identical filament-forming beams intersecting in air. A 7% amplification of one beam occurs at the energy expense of the other in a single interaction, controllable by adjusting their relative delay by tens of femtoseconds. The data are consistent with the impulsive Raman nonlinear response of the air molecules as the coupling mechanism. The filament conical emission is controllably enhanced or suppressed by the interaction, indicating that two-beam coupling may be an effective means for filament regeneration and control.

Paediatric regional anaesthesia, a survey of practice in the United Kingdom.

BACKGROUND: A variety of techniques and drugs, many unlicensed, is used in paediatric regional anaesthesia. This study is the first to survey paediatric anaesthetists about the techniques and drugs used in paediatric regional anaesthesia. The aim is to provide a record and benchmark of UK practice. METHODS: A postal questionnaire was sent to all members of the Association of Paediatric Anaesthetists residing in the UK. Information was requested on the type of hospital worked in, years of practice, paediatric anaesthesia workload, regional anaesthesia techniques used, and drugs used in regional anaesthesia. RESULTS: A total of 220 responses from 264 questionnaires (83.3%) were received. Of these respondents, 155 (70%) practised paediatric anaesthesia as more than 50% of their workload, and 10 had retired or returned blank forms. Two hundred and two of 210 (96%) use caudal anaesthesia and 151 (72%) use caudal, epidural and peripheral block. One hundred and ninety-two of 210 (91%) have no lower age limit for using caudal anaesthesia. One hundred and twenty-three of 210 anaesthetists (58%) used adjuvants with local anaesthetics in caudal block, the most common being fentanyl [44/210 (21%)], clonidine [55/210 (26%)], diamorphine [27/210 (13%)] and ketamine [67/210 (32%)]. Those working in specialist centres or teaching hospitals or who had a greater paediatric anaesthesia workload were more likely to use a greater variety of regional anaesthesia techniques. CONCLUSIONS: Caudal anaesthesia is widely used for patients of all ages by almost all practitioners. Most anaesthetists at all hospital types and experience levels use adjuvants with local anaesthetics when performing caudal anaesthesia. Those with more experience in paediatric anaesthesia and those in specialist centres commonly use other neuraxial and peripheral block techniques.

A method for UV-bonding in the fabrication of glass electrophoretic microchips.

This paper presents an approach for the development of methodologies amenable to simple and inexpensive microchip fabrication, potentially applicable to dissimilar materials bonding and chip integration. The method involves a UV-curable glue that can be used for glass microchip fabrication bonding at room temperature. This involves nothing more than fabrication of glue "guide channels" into the microchip architecture that upon exposure to the appropriate UV light source, bonds the etched plate and cover plate together. The microchip performance was verified by capillary zone electrophoresis (CZE) of small fluorescent molecules with no microchannel surface modification carried out, as well as with a DNA fragment separation following surface modification. The performance of these UV-bonded electrophoretic microchips indicates that this method may provide an alternative to high temperature bonding.

Intra- and interlaboratory variabilities of results obtained with the Quantiplex human immunodeficiency virus type 1 RNA bDNA assay, version 3.0.

Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at -70 degrees C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at < or =-70 degrees C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of > or =1,000 copies/ml at CPAL, 148 (72%) of the results varied by < or =0.20 log(10) when tested at Hershey and none varied by >0.50 log(10). However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log(10) when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log(10) while the maximum interrun variation was 0.52 log(10). High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log(10), while the maximum interrun variation was 0.55 log(10). In our patient population, a change in bDNA HIV-1 RNA results of < or =0.50 log(10) over time most likely represents normal laboratory test variation. However, a change of >0.50 log(10), especially if the results are >1,000 copies/ml, is likely to be significant.

Acousto-optical deflection-based whole channel scanning for microchip isoelectric focusing with laser-induced fluorescence detection.

This paper describes the development of a technique amenable to the separation of proteins on a microchip by isoelectric focusing (IEF) with entire channel scanning laser-induced fluorescence detection using acousto-optical deflection (AOD). The ability to use AOD to scan the portions of or the entire length of an IEF separation channel allows for high-speed analysis since the mobilization step is circumvented with this technique. Employing no moving parts eliminates mechanical noise and, not only is there no loss of resolution, AOD scanning can potentially increase resolution. The ability of AOD to provide ultra-fast scanning rates (kHz timescale) allows for real-time imaging of the focusing process. This is demonstrated with the separation of naturally fluorescent proteins using entire channel (total scanning range of 2.4 cm) AOD-mediated scanning laser-induced fluorescence detection.

Hepatitis C viral clearance and antibody reactivity patterns in persons with haemophilia and other congenital bleeding disorders.

We studied hepatitis C virus (HCV) clearance and antibody reactivity patterns in a cohort of 100 haemophiliacs exposed to unsterilized blood products, of whom 25 were antiHCV negative and 75 were antiHCV positive [49 human immunodeficiency virus (HIV) negative and 26 HIV positive]. HCV RNA was measured by the 2.0 bDNA assay and an 'in-house' polymerase chain reaction assay. Antibody reactivity patterns were examined using a recombinant immunoblot assay (RIBA). Prior HCV infection was found in two (8%) of 25 antiHCV negative patients. HCV viraemia persisted in all 26 antiHCV+ patients who were coinfected with HIV. HCV RNA clearance was found in 12 (25%) of 49 antiHCV+, HIV- patients. Viral clearance was associated with younger current age (P < 0.01) and age at infection (P < 0.001), but not with duration of infection or with dose or frequency of clotting factor use. RIBA ratios reflecting an index of each patient's overall reactivity to four HCV epitopes were significantly lower in those with viral clearance (P < 0.0001). Over a period of 15 years, those with viral clearance demonstrated significant loss of reactivity to the NS3, NS4 and NS5 epitopes, while those with viral persistence demonstrated relatively stable reactivities to all epitopes. We conclude that spontaneous HCV RNA clearance in haemophiliacs is age-related and is unlikely to occur in those coinfected with HIV. The loss of antibody reactivity for some epitopes, especially c22 (core), may be a marker for the natural resolution of chronic HCV infection.

Analysis of a large collection of natural HIV-1 integrase sequences, including those from long-term nonprogressors.

A large collection of natural HIV-1 integrase (IN) sequences has not previously been described. We reasoned that analysis of such sequences would address whether natural variation of HIV-1 IN contributes to the pathogenesis of AIDS and might also identify amino acid residues important for IN function. Sequences encoding HIV-1 IN were amplified from cryopreserved lymphocytes or plasma obtained at different times from 10 hemophilia patients who had been observed for up to 17 years. The region of the HIV-1 genome that encodes the 288-amino acid IN protein was sequenced from a total of 102 clones; information was obtained for 99.97% of 29,478 amino acid positions. Phylogenetic analysis indicated that patient samples were unique. Interpatient nucleic acid distances ranged from 0.8% to 4.9%, highlighting the tight conservation of this genomic region. No major differences were found between DNA and RNA or between early and late time points from the same patient. Significantly, no amino acid changes that might account for the variable rate of disease progression between patients were evident. Only one amino acid substitution involved a highly conserved residue known to be important for enzymatic activity. However, several interesting amino acid substitutions were noted, including residues within the C-terminal region of the protein for which sequence comparisons between animal retroviruses have not been very informative. These results should encourage the pursuit of anti-integrase therapies, especially inasmuch as the apparent biologic constraints on the IN sequence may deter the development of drug resistance.

Morphine pharmacokinetics during continuous infusion of morphine sulfate for infants receiving extracorporeal membrane oxygenation.

OBJECTIVES: To determine a) if serum morphine concentration changes during the first 3 hrs of extracorporeal membrane oxygenation (ECMO); and b) if absorption of morphine onto the membrane oxygenator is responsible for these changes. Also, morphine clearance during the first 5 days of ECMO was studied. DESIGN: Prospective, open-label study with consecutive patient enrollment. SETTING: Neonatal intensive care unit at a university-affiliated, children's hospital. SUBJECTS: Eleven neonates with severe persistent pulmonary hypertension of the newborn receiving continuous intravenous infusions of morphine sulfate and requiring ECMO. INTERVENTIONS: Blood samples were obtained from the subjects and ECMO circuits at predetermined time intervals. MEASUREMENTS AND MAIN RESULTS: Serum morphine concentration was determined using high-performance liquid chromatography. Morphine concentrations were no different from baseline at 5 mins, 1 hr, or 3 hrs after beginning ECMO. There was no significant difference in morphine concentration from samples taken immediately proximal and distal to the membrane oxygenator at 5 mins, 1 hr, and 3 hrs after the start of ECMO. Morphine clearance was calculated on days 1, 3, and 5 of ECMO. The mean value for morphine clearance was 11.7 +/- 9.3 (SD) ml/min/kg (range 2.6 to 34.5). CONCLUSIONS: The initiation of ECMO does not lead to a significant decrease in serum morphine concentration and there is no uptake of morphine onto the membrane oxygenator of the ECMO circuit. Morphine clearance for infants receiving ECMO is variable.

Suppression of hepatitis C virus (HCV) replication by hepatitis D virus (HDV) in HIV-infected hemophiliacs with chronic hepatitis B and C.

Most hemophiliacs who are coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) have high serum levels of HCV RNA. To study the impact of multiple hepatitis virus infections, we evaluated all eight chronic carriers of hepatitis B surface antigen (HBsAg) from a previously studied cohort of 99 hemophiliacs with chronic HIV and HCV infections. Stored serum or plasma samples were tested for antibody to hepatitis D virus (anti-HDV) by ELISA; qualitatively for HCV RNA, HBV DNA, and HDV RNA by the polymerase chain reaction (PCR); and quantitively for HIV RNA, HCV RNA, and hepatitis B virus (HBV) DNA by a quantitative branched DNA signal amplification assay. HCV RNA was detected in only one of five patients with HDV infections on a cross-sectional study, and this individual had low levels (< 3.5 x 10(5) genome eq/ml) of HCV RNA. In contrast, all three without HDV infections had high levels (> 1.5 x 10(7) genome eq/ml) of HCV RNA. HIV RNA was present in all eight patients. There was no correlation between the level of HIV RNA and the presence of hepatitis viruses. Three of the eight patients (38%) died of liver failure and another has hypersplenism with hypoprothrombinemia. We conclude that HDV infection appears to suppress HCV replication and that liver failure is common in adult HIV-infected hemophiliacs with chronic HCV and HBV infections. These findings have implications for the therapy of HCV-infected hemophiliacs who are HBsAg positive.

Secondary structure of M13 coat protein in phospholipids studied by circular dichroism, Raman, and Fourier transform infrared spectroscopy.

There is considerable uncertainty about the precise secondary structure adopted by the M13 coat protein when embedded in a phospholipid bilayer. Circular dichroism (CD) spectroscopy suggests that a major change in the structure of the coat protein occurs upon membrane insertion. It is reported that the structure of the protein in the membrane has only about 50% alpha-helix, the rest being mainly in a beta-sheet conformation, whereas the protein is almost completely alpha-helical when intact in the phage. In this study we have undertaken a spectroscopic analysis using Fourier transform infrared, Raman, and CD spectroscopy to characterize the secondary structure of M13 coat protein when present in membranes consisting of dioleoylphosphatidylglycerol and dimyristoylphosphatidylglycerol. In sharp contrast to earlier CD studies, our results indicate that the coat protein in its membrane-embedded state has a very high alpha-helical content with virtually no beta-sheet structures present. This result indicates that the structures of the coat protein when intact in the phage or when embedded in the membrane are similar. Although our results differ from earlier CD studies, they are consistent with a recent NMR study, which showed that the M13 coat protein in sodium dodecyl sulfate micelles is primarily alpha-helical with no evidence for beta-sheet structure [Henry, G. D., & Sykes, B.D. (1992) Biochemistry 31, 5284-5297]. These results lead to the conclusion that the M13 coat protein can insert from the membrane-bound state into a virus particle with a similar secondary structure, without large energy implications.(ABSTRACT TRUNCATED AT 250 WORDS)

The macroscopic organization of reconstituted M13 coat protein-phospholipid systems. An EPR spectroscopy and polarizing microscope study.

The coat protein of the bacteriophage M13 in the alpha-helical state is reconstituted in macroscopically oriented systems of dioleoylphosphatidylcholine that are prepared by squeezing the reconstituted material between glass plates. The coat protein dramatically influences the macroscopic orientation of the multibilayers, as is investigated by polarizing microscopy and EPR spectroscopy of the cholestane spin label embedded in the bilayers. It is found that with increasing amounts of protein the spontaneous macroscopic orientation of the reconstituted system decreases. This effect is proposed to be due to an increase of the apparent viscosity of the lipid-protein systems with increasing amounts of protein. This is assumed to arise from a sticky effect of the C- and N-terminal protein parts that extend into the aqueous phase between the bilayers.

Formation of non-bilayer structures induced by M13 coat protein depends on the conformation of the protein.

A comparison is made of the interaction of the coat protein of bacteriophage M13 in a predominant alpha-helix conformation and in a predominant beta-sheet conformation. To perform a systematic study of the interaction between the protein in these two different forms of the surrounding lipid matrix, NMR spectra of 2H-nuclei of specific labelled phospholipid systems are measured. In addition 31P-NMR is employed to provide information about the morphological structure adopted by the reconstituted lipid/protein systems. From the 2H-NMR studies on specific headgroup and chain deuterium labelled phospholipids it is found that the protein in the predominant beta-sheet conformation causes a fraction of lipids to be trapped. By combining the results from the headgroup and acyl chains of the phospholipids, it is concluded that the trapped lipids are arranged in a non-bilayer structure, probably caused by a misfitting of the hydrophobic core of the protein and the membrane bilayer. The protein in the predominant alpha-helix conformation perfectly fits in the lipid bilayer and has only minor influences on the surrounding lipid matrix. A new model is proposed to explain the presence of the trapped lipids in the lipid/protein systems.

Stoichiometry, selectivity, and exchange dynamics of lipid-protein interaction with bacteriophage M13 coat protein studied by spin label electron spin resonance. Effects of protein secondary structure.

Bacteriophage M13 major coat protein has been isolated with cholate and reconstituted in dimyristoyl- and dioleoylphosphatidylcholine (DMPC and DOPC, respectively) bilayers by dialysis. Fourier transform infrared spectra of DMPC/coat protein recombinants confirmed that, whereas the protein isolated by phenol extraction was predominantly in a beta-sheet conformation, the cholate-isolated coat protein contained a higher proportion of the alpha-helical conformation [cf. Spruijt, R. B., Wolfs, C. J. A. M., & Hemminga, M. A. (1989) Biochemistry 28, 9158-9165]. The cholate-isolated coat protein/lipid recombinants gave different electron spin resonance (ESR) spectral line shapes of incorporated lipid spin labels, as compared with those from recombinants with the phenol-extracted protein that were studied previously [Wolfs, C. J. A. M., Horváth, L. I., Marsh, D., Watts, A., & Hemminga, M. A. (1989) Biochemistry 28, 9995-10001]. Plots of the ratio of the fluid/motionally restricted components in the ESR spectra of spin-labeled phosphatidylglycerol were linear with respect to the lipid/protein ratio in the recombinants up to 20 mol/mol. The corresponding values of the relative association constants, Kr, and number of association sites, N1, on the protein were Kr approximately 1 and N1 approximately 4 for DMPC recombinants and Kr approximately 1 and N1 approximately 5 for DOPC recombinants. Simulation of the two-component lipid spin label ESR spectra with the exchange-coupled Bloch equations gave values for the off-rate of the lipids leaving the protein surface of 2.0 x 10(7) s-1 at 27 degrees C in DMPC recombinants and 3.0 x 10(7) s-1 at 24 degrees C in DOPC recombinants.(ABSTRACT TRUNCATED AT 250 WORDS)

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers.

Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the alpha-helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to beta-polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D parallel) and perpendicular (D perpendicular) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P infinity to L/P 15 results in a decrease of the rotational diffusion coefficient D perpendicular from 3.4 x 10(7) to 1.9 x 10(7) s-1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters and , and the rotational diffusion coefficient D perpendicular of the fluorescent probe. The order parameters and increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformation and aggregation of M13 coat protein studied by molecular dynamics.

Molecular dynamics (MD) simulations are performed on M13 coat protein, a small membrane protein for which both alpha- and beta-structures have been suggested. The simulations are started from initial conformations that are either monomers or dimers of alpha-helices or U-shaped beta-sheets. The lipid bilayer is represented by a hydrophobic potential. The results are analyzed in terms of stability, energy and secondary structure. The U-shaped beta-structure changes from a planar to a twisted form with larger twist for the monomer than the dimer. The beta-sheet is much more flexible than the alpha-helix as monitored by the root mean square (rms) fluctuations of the C alpha atoms. A comparison of the energies after 100 ps MD simulation shows that of the monomers, the alpha-helix has the lowest energy. The energy difference between alpha- and beta-structures decreases from 266 kJ/mol to 148 kJ/mol, when going from monomers to dimers. It is expected that this difference will decrease with higher aggregation numbers.