bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo detection of a pH‐sensitive nitroxide in the rat stomach by low‐field ESR‐based techniques
Authors: Foster Margaret A. , Grigor'ev Igor A. , Lurie David J. , Khramtsov Valery V. , McCallum Stephen , Panagiotelis Ioannis , Hutchison James M.S. , Koptioug Andrei , Nicholson Ian
Published: February 2003

A study was made of the in vivo detectability of a pH‐sensitive, imidazolidine spin probe, and the efficacy of low‐frequency electron spin resonance (ESR)‐based techniques for pH measurement in vitro and in vivo in rats. The techniques used were longitudinally‐detected ESR (LODESR) and field‐cycled dynamic nuclear polarization (FC‐DNP) for in vitro and in vivo measurements, and radiofrequency (RF)‐ and X‐band ESR for comparisons in vitro. The spin probe was hexamethyl imidazolidine (HMI) with a pK of 4.6. All techniques detected HMI. Detection by FC‐DNP implies coupling between the free radical and solvent water spins. Separations between the three spectral lines of the nitroxide radical, relative to measurement frequency, were consistent with theory. The overall spectrum width from unprotonated HMI (pH > pK) was greater than that from protonated agent (pH < pK). This was observed in vitro and in vivo. Longer‐term studies showed that HMI is detectable and has the same spectral width (i.e., is at the same pH) up to 2 hr after gavage into the stomach, although the magnitude of the signal decreases rapidly during the first hour. These findings demonstrate the suitability of LODESR and FC‐DNP for monitoring HMI and measuring pH in vivo. These techniques would be useful for monitoring disease and drug pharmacology in the living system. Magn Reson Med 49:558–567, 2003. © 2003 Wiley‐Liss, Inc.
Mechanism of magnetization transfer during on‐resonance water saturation. A new approach to detect mobile proteins, peptides, and lipids
Authors: van Zijl Peter C.M. , Zhou Jinyuan , Mori Noriko , Payen Jean‐Francois , Wilson David , Mori Susumu
Published: February 2003

The mechanism of magnetization transfer (MT) between water and components of the proton spectrum was studied ex vivo in a perfused cell system and in vivo in the rat brain (n = 5). Water was selectively labeled and spectral buildup consequential to transfer of longitudinal magnetization was followed as a function of time. At short mixing time (Tm), nitrogen‐bound solvent‐exchangeable protons were observed, predominantly assigned to amide groups of proteins and peptides. At longer Tm, intramolecular nuclear Overhauser enhancement (NOE) was observed in the aliphatic proton region, leading to a mobile‐macromolecule‐weighted spectrum that resembles typical protein spectra described in the literature. This effect on the proton spectrum is distinct from that of classical off‐resonance MT, which has been shown to be due to the immobile solid‐like proton pool. When studying a solution of major brain metabolites under physiological concentrations and conditions (pH), no transfer effects were observed, in line with expectations based on reduced NOE effects in rapidly tumbling molecules and the fast proton exchange rates of amino, amine, SH, and OH groups. The spectral intensities of the amide protons may serve as indicators for pH and cellular levels of mobile proteins and peptides, while the aliphatic components are representative of several types of mobile macromolecules, including proteins, peptides, and lipids. Magn Reson Med 49:440–449, 2003. © 2003 Wiley‐Liss, Inc.
A correlative measure for processing multiangle diffusion‐weighted images
Authors: Pipe James G. , Farthing Victoria G.
Published: February 2003

A simple metric for the shared diffusion anisotropy between two voxels is proposed. The metric has large values when the diffusion is highly anisotropic in both voxels and oriented in the same direction. This metric can be used in several ways, three of which are illustrated in this article. First, the metric can be used to rapidly calculate a fractional anisotropy measure using an arbitrary number of measured diffusion values. Second, a simple way to exploit the metric for filtering DWI data with neighboring voxels with similar diffusion characteristics is shown. Third, the metric can be used to create color images that reflect tract orientation. Magn Reson Med 49:536–542, 2003. © 2003 Wiley‐Liss, Inc.
Comparison of the return‐to‐the‐origin probability and the apparent diffusion coefficient of water as indicators of necrosis in RIF‐1 tumors
Authors: Helmer Karl G. , Meiler Michael R. , Sotak Christopher H. , Petruccelli Joseph D.
Published: February 2003

Two model‐independent measures of diffusion, the apparent diffusion coefficient (ADC) and return‐to‐the‐origin probability enhancement (R) were compared for their ability to detect tissue necrosis in RIF‐1 murine tumors. Both reflect the degree of restriction experienced by the endogenous water molecules; however, the ADC is calculated from the initial linear slope of the diffusion attenuation curve, while R is calculated from data that includes the non‐monoexponential part of the curve. In spectroscopic studies (n = 9), neither the ADC nor R showed a strong correlation with tumor volume. In imaging studies (n = 14), ADC, R, and T2 were calculated on a pixel‐by‐pixel basis. There, the mean ADC and mean R for the entire imaging slice showed reasonable correlation with necrotic tumor fraction (r2 = 0.679 and −0.665, respectively). The mean T2 value yielded a poor correlation (r2 = 0.436). Regions‐of‐interest were chosen from areas identified as either necrotic or viable and the resulting sets of ADC and R‐values were subjected to discriminant analysis to determine the identification error rate. The error was greater for R than for the ADC (P < 0.001). Therefore, in this application, the use of the non‐monoexponential part of the diffusion attenuation curve does not provide additional identification power. Magn Reson Med 49:468–478, 2003. © 2003 Wiley‐Liss, Inc.
Functional magnetic resonance imaging of the human brain based on signal enhancement by extravascular protons (SEEP fMRI)
Authors: Stroman P.W. , Tomanek B. , Krause V. , Frankenstein U.N. , Malisza K.L.
Published: February 2003

Functional magnetic resonance imaging (fMRI) studies of the human brain were carried out at 3 Tesla to investigate an fMRI contrast mechanism that does not arise from the blood oxygen‐level dependent (BOLD) effect. This contrast mechanism, signal enhancement by extravascular protons (SEEP), involves only proton‐density changes and was recently demonstrated to contribute to fMRI signal changes in the spinal cord. In the present study it is hypothesized that SEEP fMRI can be used to identify areas of neuronal activity in the brain with as much sensitivity and precision as can be achieved with BOLD fMRI. A detailed analysis of the areas of activity, signal intensity time courses, and the contrast‐to‐noise ratio (CNR), is also presented and compared with the BOLD fMRI results. Experiments were carried out with subjects performing a simple finger‐touching task, or observing an alternating checkerboard pattern. Data were acquired using a conventional BOLD fMRI method (gradient‐echo (GE) EPI, TE = 30 ms), a conventional method with reduced BOLD sensitivity (GE‐EPI, TE = 12 ms), and SEEP fMRI (spin‐echo (SE) EPI, TE = 22 ms). The results of this study demonstrate that SEEP fMRI may provide better spatial localization of areas of neuronal activity, and a higher CNR than conventional BOLD fMRI, and has the added benefit of lower sensitivity to field inhomogeneities. Magn Reson Med 49:433–439, 2003. © 2003 Wiley‐Liss, Inc.
Investigation into the wrinkling behaviour of thin sheets in the cylindrical cup deep drawing process using bifurcation theory
Authors: Kim J. B. , Yoon J. W. , Yang D. Y.
Published: February 2003

The initiation and growth of wrinkles are influenced by many factors such as stress ratios, the mechanical properties of the sheet material, the geometry of the workpiece, and contact condition. It is difficult to analyse wrinkling initiation and growth while considering all the factors because the effects of the factors are very complex and studies of wrinkling behaviour may show a wide scattering of data even for small deviations in factors. In the present study, the bifurcation theory is introduced for the finite element analysis of wrinkling initiation and growth. All the above mentioned factors are conveniently considered by the finite element method. Wrinkling initiation is found by checking the determinant of the stiffness matrix and wrinkling behaviour is analysed by successive iteration with the perturbed guess along the eigenvector.

Wrinkling initiation and growth in the cylindrical cup deep drawing process are analysed by using the proposed bifurcation algorithm. The mechanism of wrinkling initiation and growth in the cylindrical cup deep drawing process is investigated in detail. The results of the finite element analysis are compared with the experimental results. Through the finite element analyses of wrinkling growth for various material constants, the effects of plastic anisotropy and the strain hardening coefficient on the wrinkling behaviour of sheet metal are investigated. Copyright © 2003 John Wiley & Sons, Ltd.
Minimizing dynamic effects while using dynamic codes to simulate binder‐set processes
Authors: Hu Weilong
Published: February 2003

When using a dynamic code to simulate a quasi‐static job, the dynamic effect is a very serious problem, such as in the binder set simulation in stamping process design. Dynamic effects generally consist of two different acting types, oscillation due to the kinetic energy of the blank and additional deformation thanks to the inertial action. For the binder set simulation, the inertial effect is more apt to change the simulation result due to less‐constrained forming of the blank. Light effect varies the magnitude of blank deformation. Seriousness shall result in the deformation type change of the forming blank, which could make simulating results useless for real‐world application. To aim at the dynamic problems, especially in the inertial effect, several useful and practical ways have been proposed in this paper. Some parameters that we can use to minimize dynamic effects during a simulation effectively include: the velocity type of the travelling part, damping, defining the travelling part and imposing additional loads on the forming blank. Finally, four associated simulation cases have been discussed to show how these enhancing methods can be applied in real stamping production. Copyright © 2003 John Wiley & Sons, Ltd.
Iterative solution of a hybrid method for Maxwell's equations in the frequency domain
Authors: Edlund Johan , Lötstedt Per , Strand Bo
Published: February 2003

A hybrid method for solution of Maxwell's equations of electromagnetics in the frequency domain is developed as a combination between the method of moments and the approximation in physical optics. The equations are discretized by a Galerkin method and solved by an iterative block Gauss–Seidel method. The convergence of the iterations is studied theoretically and in numerical experiments. The accuracy of the hybrid method is compared to the method of moments for a cylinder with an incident field for different wavenumbers. Copyright © 2003 John Wiley & Sons, Ltd.
ε‐N,N,N‐Trimethyllysine‐specific ions in matrix‐assisted laser desorption/ionization‐tandem mass spectrometry
Authors: Hirota Junko , Satomi Yoshinori , Yoshikawa Kazuaki , Takao Toshifumi
Published: February 2003

ε‐N,N,N‐Trimethyllysine (Kme3) is a component of a number of proteins and plays an important role in the expression of their biological functions. Trimethylation, which causes an incremental increase in mass of 42.0470 Da from that of the corresponding MH+ ion, cannot be distinguished from the acetylation (+42.0106 Da), which also occurs on ε‐amino groups of Lys or α‐amino groups in many proteins, without high‐accuracy mass measurement which is accurate to within the second decimal place. MALDI‐MS and MS/MS have been applied for the analyses of post‐translational modifications of histone H3, which is known to contain both multiple acetylation and methylation sites in its sequence. During the measurements of the modified peptides, a novel fragmentation which involves the loss of trimethylamine from Kme3 was found. This characteristic fragmentation, which was observed to produce ions separated by 59 Da from the conventional precursor ion or sequence ions, would be useful for probing Kme3 units in the sequence. Copyright © 2003 John Wiley & Sons, Ltd.
Analysis of rabbit tear proteins by high‐pressure liquid chromatography/electrospray ionization mass spectrometry
Authors: Zhou Lei , Beuerman Roger W. , Barathi Amutha , Tan Donald
Published: February 2003

The aim of this study was to develop a fast and reliable analytical procedure for the display of the protein components of tears that can be used to differentiate the status of the ocular surface. Using this new procedure, we analyzed the tear protein components following a corneal wound in the rabbit. Calibrated 10‐μL glass, fire‐polished capillary micropipettes were used to collect tears from New Zealand White rabbits prior to and daily for 9 days following a unilateral 6‐mm diameter centrally placed anterior keratectomy. Tear proteins were eluted by a reversed‐phase high‐performance liquid chromatography (RP‐HPLC) column and the tear protein profile was monitored by electrospray ionization (ESI) mass spectrometry positive total ion current (TIC) chromatography. Tear proteins were reliably separated into 17 peaks, each of which contained one or a number of protein components. The molecular weight of each protein component was determined by on‐line ESI. Major tear protein components, lactoferrin, lysozyme (minimally detectable in rabbit tears), albumin, lipocalin, lipophilin and β2‐microglobulin, were tentatively identified by this method. Based on the mass spectrometric data, β2‐microglobulin was found to be glycosylated with N‐acetylhexosamine. ESI‐positive TIC chromatograms and mass spectra revealed comparative differences in the tear protein spectra after corneal wounding. One day after wounding, rabbit lysozyme with a molecular weight of 14 717 Da was found to be 8‐fold higher in the tears of wounded eyes when compared with tears from unwounded eyes. It dropped back to normal 3 days after wounding. The expression of an unidentified tear protein with the molecular weight of 16 060 Da was also elevated after corneal wounding and returned to normal level by day 5. In this study, LC/ESI‐MS was developed as a fast, reproducible and simple method for the identification and analysis of many of the protein components of the tears. Importantly, this technique also allows quantification of each component resolved in the chromatogram. This method is very suitable for mapping peptides and proteins (<80 kDa) in tears. Copyright © 2003 John Wiley & Sons, Ltd.
Automated quantitative and isotopic (13C) analysis of dissolved inorganic carbon and dissolved organic carbon in continuous‐flow using a total organic carbon analyser
Authors: St‐Jean Gilles
Published: February 2003

A method for the automated 13C analysis of dissolved inorganic and organic carbon species has been developed to operate on a continuous‐flow isotope ratio mass spectrometer (CF‐IRMS). For natural and anthropogenic carbon species, the 13C stable isotope has proven to be an excellent environmental tracer. Analytical performance tests were carried out on various organic compounds from easily oxidisable (sugar) to difficult (humic acid). A set of natural samples was also analysed to confirm the flexibility of the system. Analytical precision (2σ) is typically <0.20‰ with sample reproducibility from 0.10–0.35‰ depending on reactivity of material. We believe this to be the first successful use of a total organic carbon (TOC) analyser for both dissolved inorganic and, specifically, dissolved organic species for 13C stable isotope analysis in an automated CF‐IRMS system. Routine analysis is achieved fairly quickly, is relatively simple with little or no sample manipulation, and will allow new and exciting studies for stable isotope research in both natural abundance and organic tracer studies not easily achieved before. Copyright © 2003 John Wiley & Sons, Ltd.
Heuristic charge assignment for deconvolution of electrospray ionization mass spectra
Authors: Zheng Huiru , Ojha Piyush C. , McClean Stephen , Black Norman D. , Hughes John G. , Shaw Chris
Published: February 2003

We propose a new algorithm for deconvolution of electrospray ionization mass spectra based on direct assignment of charge to the measured signal at each mass‐to‐charge ratio (m/z). We investigate two heuristics for charge assignment: the entropy‐based heuristic is adapted from a deconvolution algorithm by Reinhold and Reinhold;10 the multiplicative‐correlation heuristic is adapted from the multiplicative‐correlation deconvolution algorithm of Hagen and Monnig.6 The entropy‐based heuristic is insensitive to overestimates of zmax, the maximum ion charge. We test the deconvolution algorithm on two single‐component samples: the measured spectrum of human beta‐endorphin has two prominent and one very weak line whereas myoglobin has a well‐developed quasi‐gaussian envelope of 17 peaks. In both cases, the deconvolution algorithm gives a clean deconvoluted spectrum with one dominant peak and very few artefacts. The relative heights of the peaks due to the parent molecules in the deconvoluted spectrum of a mixture of two peptides, which are expected to ionize with equal efficiency, give an accurate measure of their relative concentration in the sample. Copyright © 2003 John Wiley & Sons, Ltd.
Validating methods for measuring δ18O and δ13C in otoliths from freshwater fish
Authors: Guiguer K. R. R. A. , Drimmie R. , Power M.
Published: February 2003

The ability of the phosphoric acid digestion technique to extract carbon dioxide from biogenic carbonates and reliably reproduce δ18O and δ13C signatures from standard reference materials (NBS‐18, NBS‐19) was tested and shown to produce accurate, unbiased measurements of non‐biologic materials. The effects of roasting preparation methods commonly reported when analyzing biogenic carbonates were also tested in a series of experiments using reference standards and otoliths obtained from aquacultured Arctic charr and rainbow trout. Roasting had no effect on the isotope measurement of reference standards. No significant differences between mean oxygen isotope signatures from paired experiments with roasted and non‐roasted fish otoliths were found. However, otolith oxygen isotope measurements were significantly enriched in comparison to rearing water‐based measurements for both species. Agreement between expected isotopic equilibrium and measured otolith δ18O values varied as a function of roasting temperature and between species. Criteria for the selection of appropriate roasting temperatures are suggested and favour 350°C in freshwater fish where unbiased estimates of average rearing water temperatures and known differences in rearing temperatures were obtained. Carbon isotopic disequilibria were observed for both species. A mixing model analysis established differences in the percentage of metabolically derived carbon in studied otoliths, with Arctic charr deriving a greater proportion of otolith δ13C from metabolism as a result of higher metabolic rates. Copyright © 2003 John Wiley & Sons, Ltd.
Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix‐assisted laser desorption/ionisation mass spectrometry and high‐performance liquid chromatography/electrospray ionisation mass spectrometry
Authors: Cunsolo Vincenzo , Foti Salvatore , Saletti Rosaria , Gilbert Simon , Tatham Arthur S. , Shewry Peter R.
Published: February 2003

Structural studies of the high molecular weight (HMW) glutenin subunits 1Dy10 and 1Dy12 of bread wheat were conducted using matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOFMS) and reversed‐phase high‐performance liquid chromatography/electrospray ionisation mass spectrometry (RP‐HPLC/ESI‐MS). For both proteins, MALDI‐TOFMS analysis showed that the isolated fractions contained a second component with a mass about 500–540 Da lower than the major component. The testing and correction of the gene‐derived amino acid sequences of both proteins were performed by direct MALDI‐TOFMS analysis of their tryptic peptide mixture and analysis of the digests was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimising the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP‐HPLC/ESI‐MS analysis of the tryptic digest. This resulted in the coverage of the whole protein sequences except for two short fragments (T1 and T8), which are identical in the two homologous subunits, and for an additional dipeptide (T14) in subunit 1Dy12, which were not detected. It also demonstrated that, in contrast to the gene‐derived data, the sequence of subunit 1Dy12 does not include the dipeptide Gly‐Gln between residues Gln454 and Pro455, and that the lower mass components present in both fractions correspond to the same sequences lacking short peptides that are probably lost from the protein N‐ or C‐termini. Finally, the results obtained provide evidence for the lack of a substantial level of glycosylation or other post‐translational modifications of the two subunits, and demonstrate that mass spectrometric mapping is the most useful method presently available for the direct verification of the gene‐derived sequences of HMW glutenin subunits and similar proteins. Copyright © 2003 John Wiley & Sons, Ltd.
Application of tandem mass spectrometry combined with gas chromatography and headspace solid‐phase dynamic extraction for the determination of drugs of abuse in hair samples
Authors: Lachenmeier Dirk W. , Kroener Lars , Musshoff Frank , Madea Burkhard
Published: February 2003

A new method combination, headspace solid‐phase dynamic extraction coupled with gas chromatography/tandem mass spectrometry (HS‐SPDE/GC/MS/MS), is introduced to determine drugs of abuse in hair samples. This highly automated procedure utilizes SPDE for pre‐concentration and on‐coating derivatization as well as GC and triple quadrupole MS/MS for selective and sensitive detection. All these steps, apart from washing and cutting of the hair samples, are performed without manual intervention on a robot‐like autosampler.

SPDE is a solventless extraction technique related to solid‐phase microextraction (SPME). The analytes are absorbed from the sample headspace directly into a hollow needle with an internal coating of polydimethylsiloxane by repeated aspirate/dispense cycles.

The HS‐SPDE/GC/MS/MS procedure was applied to the analysis of methadone, the trimethylsilyl derivatives of cannabinoids and the trifluoroacetyl derivatives of amphetamines and designer drugs. The method was shown to be sensitive with detection limits between 6 and 52 pg/mg hair matrix and precision between 0.4 and 7.8% by the use of an internal standard technique. Linearity was obtained from 0.1–20 ng/mg with coefficients of correlation between 0.995 and 0.999.

Compared with conventional methods of hair analysis, HS‐SPDE/GC/MS/MS is easier to use, substantially faster, with the degree of sensitivity and reproducibility demanded in clinical and forensic toxicology. The main advantage of the SPDE technique in relation to SPME is the robustness of the capillary. Copyright © 2003 John Wiley & Sons, Ltd.
Generalized monotonic regression using random change points
Authors: Holmes C. C. , Heard N. A.
Published: February 2003

We introduce a procedure for generalized monotonic curve fitting that is based on a Bayesian analysis of the isotonic regression model. Conventional isotonic regression fits monotonically increasing step functions to data. In our approach we treat the number and location of the steps as random. For each step level we adopt the conjugate prior to the sampling distribution of the data as if the curve was unconstrained. We then propose to use Markov chain Monte Carlo simulation to draw samples from the unconstrained model space and retain only those samples for which the monotonic constraint holds. The proportion of the samples collected for which the constraint holds can be used to provide a value for the weight of evidence in terms of Bayes factors for monotonicity given the data. Using the samples, probability statements can be made about other quantities of interest such as the number of change points in the data and posterior distributions on the location of the change points can be provided. The method is illustrated throughout by a reanalysis of the leukaemia data studied by Schell and Singh. Copyright © 2003 John Wiley & Sons, Ltd.
Comparison of HIV‐1 and HIV‐2 infectivity from a prospective cohort study in Senegal
Authors: Gilbert Peter B. , McKeague Ian W. , Eisen Geoffrey , Mullins Christopher , Guéye‐NDiaye Aissatou , Mboup Souleymane , Kanki Phyllis J.
Published: February 2003

From a prospective cohort study of 1948 initially human immunodeficiency virus (HIV) uninfected female commercial sex workers followed between 1985 and 1999 in Dakar, Senegal, the authors compared the male to female per infectious sexual exposure transmission probability of HIV types one (HIV‐1) and two (HIV‐2). New non‐parametric competing risks failure time methods were used, which minimized modelling assumptions and controlled for risk factors for HIV infection. The HIV‐1 versus HIV‐2 infectivity ratio over time was estimated by the ratio of smoothed non‐parametric kernel estimates of the HIV‐1 and HIV‐2 infection hazard functions in sex workers, adjusted by an estimate of the relative HIV‐1 versus HIV‐2 prevalence in the partner population. HIV‐1 was found to be significantly more infectious than HIV‐2 throughout the follow‐up period (P < 0.001). The HIV‐1/HIV‐2 infectivity ratio was inferred to be approximately constant over time, with estimated common value 3.55. The finding of greater HIV‐1 infectivity persisted in sensitivity analyses and in covariate‐adjusted analyses, with adjusted infectivity ratio estimates ranging between 3.40 and 3.86. Understanding the mechanisms by which HIV‐1 infects more efficiently than HIV‐2 may be useful in the development of HIV‐1 vaccines. Additionally, the methodology developed here may be useful for analysing other data sets. Copyright © 2003 John Wiley & Sons, Ltd.
Missing data in the 2×2 table: patterns and likelihood‐based analysis for cross‐sectional studies with supplemental sampling
Authors: Lyles Robert H. , Allen Andrew S.
Published: February 2003

Standard measures of crude association in the context of a cross‐sectional study are the risk difference, relative risk and odds ratio as derived from a 2× 2 table. Most such studies are subject to missing data on disease, exposure, or both, introducing bias into the usual complete‐case analysis. We describe several scenarios distinguished by the manner in which missing data arise, and for each we adjust the natural multinomial likelihood to properly account for missing data. The situations presented allow for increasing levels of generality with regard to the missing data mechanism. The final case, quite conceivable in epidemiologic studies, assumes that the probability of missing exposure depends on true exposure and disease status, as well as upon whether disease status is missing (and conversely for the probability of missing disease information). When parameters relating to the missing data process are inestimable without strong assumptions, we propose maximum likelihood analysis subsequent to collecting supplemental data in the spirit of a validation study. Analytical results give insight into the bias inherent in complete‐case analysis for each scenario, and numerical results illustrate the performance of likelihood‐based point and interval estimates in the most general case. Adjustment for potential confounders via stratified analysis is also discussed. Copyright © 2003 John Wiley & Sons, Ltd.
Estimating the probability of toxicity at the target dose following an up‐and‐down design
Authors: Stylianou Mario , Proschan Michael , Flournoy Nancy
Published: February 2003

One of the most important aspects of a phase I trial or other acute toxicity study is estimating accurately the probability of toxicity that is associated with the recommended dose. We use the biased coin up‐and‐down design to allocate and isotonic regression to estimate toxicity probabilities and determine the recommended dose. We then derive, using bootstrap methods, an estimate of the probability of toxicity at the recommended dose. Small sample properties of this estimator are also evaluated. Published in 2003 by John Wiley & Sons, Ltd.
STAT3 deletion during hematopoiesis causes Crohn's disease-like pathogenesis and lethality: A critical role of STAT3 in innate immunity
Authors: Welte Thomas , Zhang Samuel S. M. , Wang Tian , Zhang Zhiyuan , Hesslein David G. T. , Yin Zhinan , Kano Arihiro , Iwamoto Yoshiki , Li En , Craft Joseph E. , Bothwell Alfred L. M. , Fikrig Erol , Koni Pandelakis A. , Flavell Richard A. , Fu Xin-Yuan
Published: February 2003

Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional mediator for many cytokines and is essential for normal embryonic development. We have generated a unique strain of mice with tissue-specific disruption of STAT3 in bone marrow cells during hematopoiesis. This specific STAT3 deletion causes death of these mice within 4–6 weeks after birth with Crohn's disease-like pathogenesis in both the small and large intestine, including segmental inflammatory cell infiltration, ulceration, bowel wall thickening, and granuloma formation. Deletion of STAT3 causes significantly increased cell autonomous proliferation of cells of the myeloid lineage, bothin vivoandin vitro. Most importantly, Stat3 deletion during hematopoiesis causes overly pseudoactivated innate immune responses. Although inflammatory cytokines, including tumor necrosis factor α and IFN-γ, are overly produced in these mice, the NAPDH oxidase activity, which is involved in antimicrobial and innate immune responses, is inhibited. The signaling responses to lipopolysaccharide are changed in the absence of STAT3, leading to enhanced NF-κB activation. Our results suggest a model in which STAT3 has critical roles in the development and regulation of innate immunity, and deletion of STAT3 during hematopoiesis results in abnormalities in myeloid cells and causes Crohn's disease-like pathogenesis.
Using temporal context to improve biosurveillance
Authors: Reis Ben Y. , Pagano Marcello , Mandl Kenneth D.
Published: February 2003

Current efforts to detect covert bioterrorist attacks from increases in hospital visit rates are plagued by the unpredictable nature of these rates. Although many current systems evaluate hospital visit data 1 day at a time, we investigate evaluating multiple days at once to lessen the effects of this unpredictability and to improve both the timeliness and sensitivity of detection. To test this approach, we introduce simulated disease outbreaks of varying shapes, magnitudes, and durations into 10 years of historical daily visit data from a major tertiary-care metropolitan teaching hospital. We then investigate the effectiveness of using multiday temporal filters for detecting these simulated outbreaks within the noisy environment of the historical visit data. Our results show that compared with the standard 1-day approach, the multiday detection approach significantly increases detection sensitivity and decreases latency while maintaining a high specificity. We conclude that current biosurveillance systems should incorporate a wider temporal context to improve their effectiveness. Furthermore, for increased robustness and performance, hybrid systems should be developed to capitalize on the complementary strengths of different types of temporal filters.
Drosophilapigmentation evolution: Divergent genotypes underlying convergent phenotypes
Authors: Wittkopp Patricia J. , Williams Barry L. , Selegue Jayne E. , Carroll Sean B.
Published: February 2003

Similar phenotypic changes have evolved independently in many animal taxa. It is unknown whether independent changes involve the same or different developmental and genetic mechanisms. Myriad pigment patterns in the genusDrosophilaoffer numerous opportunities to address this question. Previous studies identified regulatory and structural genes involved in the development and diversification of pigmentation in selected species. Here, we examineDrosophila americanaandDrosophila novamexicana, interfertile species that have evolved dramatic pigmentation differences during the few million years since their divergence. Interspecific genetic analysis was used to investigate the contribution of five specific candidate genes and other genomic regions to phenotypic divergence by testing for associations between molecular markers and pigmentation. At least four distinct genomic regions contributed to pigmentation differences, one of which included theebonygene. Ebony protein was expressed at higher levels in the more yellowD. novamexicanathan the heavily melanizedD. americana. Because Ebony promotes yellow pigment formation and suppresses melanization, the expression difference and genetic association suggest that evolution at theebonylocus contributed to pigmentation divergence betweenD. americanaandD. novamexicana. Surprisingly, no genetic association with theyellowlocus was detected in this study, and Yellow expression was identical in the two species. Evolution at theyellowlocus underlies pigmentation divergence among otherDrosophilaspecies; thus, similar pigment patterns have evolved through regulatory changes in different genes in different lineages. These findings bear upon understanding classic models of melanism and mimicry.
Multiple routes to memory: Distinct medial temporal lobe processes build item and source memories
Authors: Davachi Lila , Mitchell Jason P. , Wagner Anthony D.
Published: February 2003

A central function of memory is to permit an organism to distinguish between stimuli that have been previously encountered and those that are novel. Although the medial temporal lobe (which includes the hippocampus and surrounding perirhinal, parahippocampal, and entorhinal cortices) is known to be crucial for recognition memory, controversy remains regarding how the specific subregions within the medial temporal lobe contribute to recognition. We used event-related functional MRI to examine the relation between activation in distinct medial temporal lobe subregions during memory formation and the ability (i) to later recognize an item as previously encountered (item recognition) and (ii) to later recollect specific contextual details about the prior encounter (source recollection). Encoding activation in hippocampus and in posterior parahippocampal cortex predicted later source recollection, but was uncorrelated with item recognition. In contrast, encoding activation in perirhinal cortex predicted later item recognition, but not subsequent source recollection. These outcomes suggest that the subregions within the medial temporal lobe subserve distinct, but complementary, learning mechanisms.
Boosting immunity by antiviral drug therapy: A simple relationship among timing, efficacy, and success
Authors: Komarova Natalia L. , Barnes Eleanor , Klenerman Paul , Wodarz Dominik
Published: February 2003

Drug therapies against persistent human infections such as hepatitis C virus, hepatitis B virus, and HIV fail to consistently eradicate the infection from the host. Hence, recent emphasis has shifted to the study of antiviral therapy aimed at boosting specific immune responses. It was argued that structured therapy interruptions were required to achieve this, because such regimes have shown promising results in early HIV infection. Using mathematical models, we show that, contrary to this notion, a single phase of drug therapy can result in the establishment of sustained immunity. We present a simple relationship between timing of therapy and efficacy of the drugs required for success. In the presence of strong viral suppression, we show that therapy should be stopped relatively early, and that a longer duration of treatment leads to failure. On the other hand, in the presence of weaker viral suppression, stopping treatment too early is detrimental, and therapy has to be continued beyond a time threshold. We discuss our modeling results primarily in the context of HCV therapy during chronic infection. Although the therapy regimes explored here also have implications for HIV, virus-mediated destruction of specific immune cells renders success unlikely during the chronic phase of the infection.
Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor
Authors: Anders M. , Hansen R. , Ding R.-X. , Rauen K. A. , Bissell M. J. , Korn W. Michael
Published: February 2003

The human coxsackievirus and adenovirus receptor (CAR) represents the primary cellular site of adenovirus attachment during infection. An understanding of the mechanisms regulating its expression could contribute to improving efficacy and safety of adenovirus-based therapies. We characterized regulation of CAR expression in a 3D cell culture model of human breast cancer progression, which mimics aspects of the physiological tissue context
in vitro.
Phenotypically normal breast epithelial cells (S1) and their malignant derivative (T4-2 cells) were grown either on tissue culture plastic (2D) or 3D cultures in basement membrane matrix. S1 cells grown in 3D showed low levels of CAR, which was expressed mainly at cell–cell junctions. In contrast, T4-2 cells expressed high levels of CAR, which was mainly in the cytoplasm. When signaling through the epidermal growth factor receptor was inhibited in T4-2 cells, cells reverted to a normal phenotype, CAR protein expression was significantly reduced, and the protein relocalized to cell–cell junctions. Growth of S1 cells as 2D cultures or in 3D in collagen-I, a nonphysiological microenvironment for these cells, led to up-regulation of CAR to levels similar to those in T4-2 cells, independently of cellular growth rates. Thus, expression of CAR depends on the integrity and polarity of the 3D organization of epithelial cells. Disruption of this organization by changes in the microenvironment, including malignant transformation, leads to up-regulation of CAR, thus enhancing the cell's susceptibility to adenovirus infection.
Regulation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and mitochondrial function by MEF2 and HDAC5
Authors: Czubryt Michael P. , McAnally John , Fishman Glenn I. , Olson Eric N.
Published: February 2003

The myocyte enhancer factor-2 (MEF2) transcription factor regulates muscle development and calcium-dependent gene expression. MEF2 activity is repressed by class II histone deacetylases (HDACs), which dissociate from MEF2 when phosphorylated on two serine residues in response to calcium signaling. To explore the potential importance of MEF2/HDAC interactions in the heart, we generated transgenic mice expressing a signal-resistant form of HDAC5 under cardiac-specific and doxycycline-inducible regulation. Transgene expression resulted in sudden death in male mice accompanied by loss and morphologic changes of cardiac mitochondria and down-regulation of mitochondrial enzymes. The transcriptional coactivator PGC-1α, a master regulator of mitochondrial biogenesis and fatty acid oxidation, was also down-regulated in response to HDAC5 expression. Examination of the
PGC-1
α promoter revealed two MEF2-binding sites that mediate transcriptional activation by MEF2 and repression by HDAC5. These findings identify
PGC-1
α as a key target of the MEF2/HDAC regulatory pathway and demonstrate this pathway's importance in maintenance of cardiac mitochondrial function.
Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P
Authors: Kovrigina Elizaveta , Wesolowski Donna , Altman Sidney
Published: February 2003

The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited.
Complete genome sequence of
Lactobacillus plantarum
WCFS1
Authors: Kleerebezem Michiel , Boekhorst Jos , van Kranenburg Richard , Molenaar Douwe , Kuipers Oscar P. , Leer Rob , Tarchini Renato , Peters Sander A. , Sandbrink Hans M. , Fiers Mark W. E. J. , Stiekema Willem , Lankhorst René M. Klein , Bron Peter A. , Hoffer Sally M. , Groot Masja N. Nierop , Kerkhoven Robert , de Vries Maaike , Ursing Björn , de Vos Willem M. , Siezen Roland J.
Published: February 2003

The 3,308,274-bp sequence of the chromosome of
Lactobacillus plantarum
strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of
L. plantarum
as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover,
L. plantarum
encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation.
L. plantarum
is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of
L. plantarum
with its environment, form a lifestyle adaptation region in the chromosome.
Defective presentation of the CD1d1-restricted natural Va14Ja18 NKT lymphocyte antigen caused by β-d-glucosylceramide synthase deficiency
Authors: Stanic Aleksandar K. , De Silva A. Dharshan , Park Jang-June , Sriram Venkataraman , Ichikawa Shinichi , Hirabyashi Yoshio , Hayakawa Kyoko , Van Kaer Luc , Brutkiewicz Randy R. , Joyce Sebastian
Published: February 2003

Va14Ja18 natural T (NKT) cells play an immunoregulatory role, which is controlled by a self glycolipid(s) presented by CD1d. Although the synthetic antigen α-D-galactosylceramide (α-D-GalCer) stimulates all Va14Ja18 NKT cells, α-anomeric D-glycosylceramides are currently unknown in mammals. We have used β-D-GalCer-deficient mice and β-D-glucosylceramide (β-D-GlcCer)-deficient cells to define the chemical nature of a natural NKT cell antigen. β-D-GalCer-deficient mice exhibit normal NKT cell development and function, and cells from these animals potently stimulate NKT hybridomas. In striking contrast, the same hybridomas fail to react to CD1d1 expressed by a β-D-GlcCer-deficient cell line. Importantly, human β-D-GlcCer synthase cDNA transfer, and hence the biosynthesis of β-D-GlcCer, restores the recognition of mutant cells expressing CD1d1 by the Va14Ja18 NKT hybridomas. Additionally, suppression of β-D-GlcCer synthesis inhibits antigen presentation to Va14Ja18 NKT cells. The possibility that β-D-GlcCer itself is the natural NKT cell antigen was excluded because it was unable to activate NKT hybridomas in a cell-free antigen-presentation assay. These findings suggest that β-D-GlcCer may play an important role in generating and/or loading a natural Va14Ja18 NKT antigen.
Crystal structure of the plasmid maintenance system ɛ/ζ: Functional mechanism of toxin ζ and inactivation by ɛ
2
ζ
2
complex formation
Authors: Meinhart Anton , Alonso Juan C. , Sträter Norbert , Saenger Wolfram
Published: February 2003

Programmed cell death in prokaryotes is frequently found as postsegregational killing. It relies on antitoxin/toxin systems that secure stable inheritance of low and medium copy number plasmids during cell division and kill cells that have lost the plasmid. The broad-host-range, low-copy-number plasmid pSM19035 from
Streptococcus pyogenes
carries the genes encoding the antitoxin/toxin system ɛ/ζ and antibiotic resistance proteins, among others. The crystal structure of the biologically nontoxic ɛ
2
ζ
2
protein complex at a 1.95-Å resolution and site-directed mutagenesis showed that free ζ acts as phosphotransferase by using ATP/GTP. In ɛ
2
ζ
2
, the toxin ζ is inactivated because the N-terminal helix of the antitoxin ɛ blocks the ATP/GTP-binding site. To our knowledge, this is the first prokaryotic postsegregational killing system that has been entirely structurally characterized.
Gray-scale photolithography using microfluidic photomasks
Authors: Chen Chihchen , Hirdes Danny , Folch Albert
Published: February 2003

The ability to produce three-dimensional (3D) microstructures is of increasing importance in the miniaturization of mechanical or fluidic devices, optical elements, self-assembling components, and tissue-engineering scaffolds, among others. Traditional photolithography, the most widely used process for microdevice fabrication, is ill-suited for 3D fabrication, because it is based on the illumination of a photosensitive layer through a “photomask” (a transparent plate that contains opaque, unalterable solid-state features), which inevitably results in features of uniform height. We have devised photomasks in which the light-absorbing features are made of fluids. Unlike in conventional photomasks, the opacity of the photomask features can be tailored to an arbitrary number of gray-scale levels, and their spatial pattern can be reconfigured in the time scale of seconds. Here we demonstrate the inexpensive fabrication of photoresist patterns that contain features of multiple and/or smoothly varying heights. For a given microfluidic photomask, the developed photoresist pattern can be predicted as a function of the dye concentrations and photomask dimensions. For selected applications, microfluidic photomasks offer a low-cost alternative to present gray-scale photolithography approaches.