Insensitive Technique Research Articles

Removing acoustic ringing baseline curvature in 13 C NMR spectra for quantitative analyses.

13 C nuclear magnetic resonance (NMR) is traditionally considered an insensitive technique, requiring long acquisition times to measure dilute functionalities on large polymers. With the introduction of cryoprobes and better electronics, sensitivity has improved in a way that allows measurements to take less than 1/20th the time that they previously did. Unfortunately, a high Q-factor with cryoprobes creates baseline curvature related to acoustic ringing that affects quantitative NMR analyses. Manual baseline correction is commonly used to compensate for the baseline roll, but it is a time-intensive process. The outcome of manual baseline correction can vary depending on processing parameters, especially for complicated spectra. Additionally, it can be challenging to distinguish between broad peaks and baseline rolls. A new anti-ring pulse sequence (zgig_pisp) was previously reported to improve on the incumbent single pulse experiment (zgig). The original report presented limited comparison data with 13 C NMR, but a thorough validation is needed before broader implementation can be considered. In this work, we report the round-robin testing and comparison of zgig_pisp and zgig pulse sequences. During the testing phase, we found that zgig_pisp is practically equivalent to zgig to ±2% for the majority of integrals examined. Additionally, a short broadband inversion pulse (BIP) was demonstrated as an alternative to the originally reported adiabatic CHIRP shaped pulse. The zgig_pisp pulse sequence code for Bruker spectrometers is also simplified.

DESPERATE: A Python library for processing and denoising NMR spectra

NMR spectroscopy is an inherently insensitive technique with respect to the amount of observable signal. A common element in all NMR spectra is random thermal noise that is often characterized by a signal-to-noise ratio (SNR). SNR can be generically improved experimentally with repetitive signal averaging or during post-processing with apodization; the former of which often results in long experimental times and the latter results in the loss of spectral resolution. Denoising techniques can instead be used during post-processing to enhance SNR without compromising resolution. The most common approach relies on the singular-value decomposition (SVD) to discard noisy components of NMR data. SVD-based approaches work well, such as Cadzow and PCA, but are computationally expensive when used for large datasets that are often encountered in NMR (e.g., Carr-Purcell/Meiboom-Gill and nD datasets). Herein, we describe the implementation of a new wavelet transform (WT) routine for the fast and robust denoising of 1D and 2D NMR spectra. Several simulated and experimental datasets are denoised with both SVD-based Cadzow or PCA and WT’s, and the resulting SNR enhancements and spectral uniformity are compared. WT denoising offers similar and improved denoising compared with SVD and operates faster by several orders-of-magnitude in some cases. All denoising and processing routines used in this work are included in a free and open-source Python library called DESPERATE.

Field-cycling NMR and DNP – A friendship with benefits

Field-cycling relaxometry, or rather its electronic version with a resistive magnet which requires signal detection at a field strength of 1 Tesla or below, remains an inherently insensitive technique due to the construction compromise that goes along with the need for a fast-switching, low-inductance magnet. For the same reasons, signal lifetime is short and frequency resolution is typically not given, at least for the predominantly used hydrogen nuclei. Dynamic Nuclear Polarization (DNP) bears the potential to circumvent these disadvantages: not only has it been demonstrated to enhance magnetization by up to three orders of magnitude beyond its thermal value, but it also provides the possibility to address particular parts of a molecule, thus generating selectivity even in the absence of spectral resolution. At the same time, DNP requires the introduction of stable radicals giving rise to additional relaxation contributions. This article presents a straightforward way to recover the native relaxation rates of the undisturbed system, and shows examples in different research fields where field-cycling relaxometry is traditionally used for refining models of molecular dynamics and interactions.

Sensitivity-Enhanced 207Pb Solid-State NMR Spectroscopy for the Rapid, Non-Destructive Characterization of Organolead Halide Perovskites

Organolead halide and mixed halide perovskites (CH3NH3PbX3, CH3NH3PbX3–nYn, X and Y = Cl–, Br–, or I–) are promising materials for photovoltaics and optoelectronic devices. 207Pb solid-state NMR spectroscopy has previously been applied to characterize phase segregation and halide ion speciation in mixed halide perovskites. However, NMR spectroscopy is an insensitive technique that often requires large sample volumes and long signal averaging periods. This is especially true for mixed halide perovskites, which give rise to extremely broad 207Pb solid-state NMR spectra. Here, we quantitatively compare the sensitivity of the various solid-state NMR techniques on pure and mixed halide organolead perovskites and demonstrate that both fast MAS and DNP can provide substantial gains in NMR sensitivity for these materials. With fast MAS and proton detection, high signal-to-noise ratio two-dimensional (2D) 207Pb–1H heteronuclear correlation (HETCOR) NMR spectra can be acquired in less than half an hour from only ca...

A quasi-optical and corrugated waveguide microwave transmission system for simultaneous dynamic nuclear polarization NMR on two separate 14.1 T spectrometers

Nuclear magnetic resonance (NMR) is an intrinsically insensitive technique, with Boltzmann distributions of nuclear spin states on the order of parts per million in conventional magnetic fields. To overcome this limitation, dynamic nuclear polarization (DNP) can be used to gain up to three orders of magnitude in signal enhancement, which can decrease experimental time by up to six orders of magnitude. In DNP experiments, nuclear spin polarization is enhanced by transferring the relatively larger electron polarization to NMR active nuclei via microwave irradiation. Here, we describe the design and performance of a quasi-optical system enabling the use of a single 395 GHz gyrotron microwave source to simultaneously perform DNP experiments on two different 14.1 T (1H 600 MHz) NMR spectrometers: one configured for magic angle spinning (MAS) solid state NMR; the other configured for solution state NMR experiments. In particular, we describe how the high power microwave beam is split, transmitted, and manipulated between the two spectrometers. A 13C enhancement of 128 is achieved via the cross effect for alanine, using the nitroxide biradical AMUPol, under MAS-DNP conditions at 110 K, while a 31P enhancement of 160 is achieved via the Overhauser effect for triphenylphosphine using the monoradical BDPA under solution NMR conditions at room temperature. The latter result is the first demonstration of Overhauser DNP in the solution state at a field of 14.1 T (1H 600 MHz). Moreover these results have been produced with large sample volumes (∼100 µL, i.e. 3 mm diameter NMR tubes).

Intraoperative sentinel lymph node evaluation: Optimizing surgical pathology practices in an era of changing clinical management.

Axillary lymph node status is an independent prognostic indicator in breast cancer. Intraoperative identification of metastatic carcinoma in sentinel lymph nodes may allow for concurrent axillary lymph node dissection at the time of primary tumor excision. A retrospective review of patients undergoing primary breast cancer excision with sentinel lymph node sampling was performed. Sensitivity and specificity of imprint cytology (touch prep) with and without the incorporation of gross evaluation was determined using permanent section results as the gold standard. Five hundred sixteen lymph nodes were analyzed by imprint cytology in 213 patients, and 203 lymph nodes were analyzed in 74 patients incorporating gross examination. Sensitivity and specificity for the detection of macrometastases by touch prep alone were 60% and 99% respectively with 4 patients undergoing same-day axillary dissection for only micrometastatic disease. False negative causes included lack of transfer of malignant cells in 8 cases and misinterpretation of tumor cells in 6 cases. Incorporating gross examination in the modified protocol resulted in reduced sensitivity of 38%, but achieved the desired 100% specificity and positive predictive value. Imprint cytology alone did not reliably distinguish between micro- and macrometastatic disease. Gross assessment combined with imprint cytology allows for improved assessment of volume of axillary disease, but is an insensitive technique.

Free-Space Time Domain Position Insensitive Technique for Simultaneous Measurement of Complex Permittivity and Thickness of Lossy Dielectric Samples

A novel time domain measurement technique is proposed to facilitate the simultaneous measurement of electrical properties (complex relative permittivity) and geometrical parameters (thickness) of the material under test (MUT). The overall process is noninvasive and noncontacting, which uses the measured scattering data of the MUT in the equivalent time domain or spatial domain. The effective time domain scattering data are employed to detect the primary and secondary peaks of the overall reflection and transmission coefficients. To this end, a novel algorithm is proposed to obtain the complex permittivity and thickness of the MUT in terms of extracted reflection and transmission power peaks. From the practical point of view, the main advantage of the proposed scheme is that one avoids the complicated calibration procedure normally required to define the reference plane. For increasing the accuracy of the overall reconstruction process, an automated optimization procedure based on parameter sensitivity analysis is proposed, which uses standard time gating procedure to implement the corresponding direct problem. The proposed technique is validated by extracting the relative permittivity, the dielectric loss (effective conductivity), and the thickness of various standard materials, such as polyethylene, Plexiglas, PVC, mortar, nylon, and so on, and comparing the extracted data with their values available in the literature.

Abstract TP47: Inverse PWI/DWI Mismatch is a Typical Feature of Acute Subcortical Ischemic Stroke

Introduction / Hypothesis: Diffusion/perfusion mismatch (MM) is commonly used to assess tissue at risk in stroke patients. In small subcortical infarcts (SSI) the reverse pattern i.e. “inverse mismatch” (IM) has been shown [1]. We aimed to evaluate the frequency and characteristics of IM in a larger cohort using two different perfusion diffusion analysis approaches. Methods: We enrolled 129 patients with SSI within 24 hours of symptom onset (1000+ study, NCT00715533) comprising 44 infarcts of the thalamus, 9 located juxtacortically, 58 in the anterior choroidal artery, 18 in the lenticulostriate territory. Lesion volumes were assessed on DWI images using AnToNIa software. To evaluate perfusion maps a time-shift insensitive technique using a block-circulant matrix for deconvolution (oSVD) [2] was employed. Perfusion lesions were evaluated on MTT and Tmax (delay >2s and >6s) maps. A perfusion/diffusion ratio of 0.7 was used to define IM and 1.3 to define mismatch (MM). Results: Results are summarized in table1. For all types of data analysis and all time points, IM was the by far most frequent pathophysiological pattern. Obviously, IM was seen most often at Tmax>6s, but it was still clearly observed with looser thresholds (Tmax>2s, MTT). At earlier time of onset IM occurred less often, but was still the most frequent pattern (p=0.0026). No significant association between infarct location and perfusion pattern was observed Conclusion: IM is the most common pathophysiological constellation in SSI. We hypothesize that this is due to a lack of collateral circulation in these “endstream infarcts” and that the diffusion changes in areas without perfusion deficit reflect cytotoxic mechanisms. 1. Doege CA et al. AJNR 2003;24:1355-63 2. Wu O et al. Magn Reson Med 2003;50:164-174

A Novel Tri-Enzyme System in Combination with Laser-Driven NMR Enables Efficient Nuclear Polarization of Biomolecules in Solution

NMR is an extremely powerful, yet insensitive technique. Many available nuclear polarization methods that address sensitivity are not directly applicable to low-concentration biomolecules in liquids and are often too invasive. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is no exception. It needs high-power laser irradiation, which often leads to sample degradation, and photosensitizer reduction. Here, we introduce a novel tri-enzyme system that significantly overcomes the above challenges, rendering photo-CIDNP a practically applicable technique for NMR sensitivity enhancement in solution. The specificity of the nitrate reductase (NR) enzyme is exploited to selectively in situ reoxidize the reduced photo-CIDNP dye FMNH2. At the same time, the oxygen-scavenging ability of glucose oxidase (GO) and catalase (CAT) is synergistically employed to prevent sample photodegradation. The resulting tri-enzyme system (NR-GO-CAT) enables prolonged sensitivity-enhanced data collection in 1D and 2D heteronuclear NMR, leading to the highest photo-CIDNP sensitivity enhancement (48-fold relative to SE-HSQC) achieved to date for amino acids and polypeptides in solution. NR-GO-CAT extends the concentration limit of photo-CIDNP NMR down to the low micromolar range. In addition, sensitivity (relative to the reference SE-HSQC) is found to be inversely proportional to sample concentration, paving the way for the future analysis of even more diluted samples.

In Vivo Clinical Measures of Intermediary Metabolism Are Inadequate

The heart derives most of its energy via mitochondrial oxidative metabolism. The heart's fuels or energy substrates include fatty acids, lactate, glucose, and ketones. Substrate concentrations vary during the day, driven mainly by postprandial status. Entry into mitochondrial oxidation requires metabolism to acetyl-coenzyme A (CoA), the fuel of the tricarboxylic acid (TCA) cycle. Acetyl-CoA oxidation generates reduced Nicotinamide adenine dinucleotide (NADH), which drives oxidative phosphorylation, the reduction of O2 to H2O, and, ultimately, the synthesis of ATP, which is essential for myocardial mechanics. The heart uses this highly regulated series of enzyme-catalyzed reactions to convert chemical energy into mechanical energy.1 Metabolism and function in the heart are inextricably linked. Article see p 201 The pathological states of the heart, such as ischemia, hypertrophy, and failure, modify metabolism. Available technologies to measure intermediary metabolism have been inadequate. Magnetic resonance spectroscopy (MRS) can be used to spatially map or at least locally measure metabolism in vivo. Measurement of endogenous triglycerides in patients using 1H MRS has demonstrated that cardiac steatosis precedes the onset of type 2 diabetes mellitus and left ventricular systolic dysfunction.2 In the failing human heart and in preclinical heart failure models, 31P MRS has shown that flux through the creatinine kinase reaction is altered.3,4 Also, ATP and phosphocreatine are reduced after myocardial infarction due to myocyte loss.5 Myocardial energy substrate can be measured with 13C MRS. It has the chemical specificity, but its inherently low sensitivity limits the spatial and temporal resolution necessary for clinical application. In isolated hearts, 13C MRS has to be used with labeled substrate molecules to define TCA cycle kinetics. The metabolites of the TCA cycle have not been measured directly in these experiments. The content of the TCA cycle metabolites is …

1H-Detected 13C Photo-CIDNP as a Sensitivity Enhancement Tool in Solution NMR

NMR is a powerful yet intrinsically insensitive technique. The applicability of NMR to chemical and biological systems would be substantially extended by new approaches going beyond current signal-to-noise capabilities. Here, we exploit the large enhancements arising from (13)C photochemically induced dynamic nuclear polarization ((13)C photo-CIDNP) in solution to improve biomolecular NMR sensitivity in the context of heteronuclear correlation spectroscopy. The (13)C-PRINT pulse sequence presented here involves an initial (13)C nuclear spin polarization via photo-CIDNP followed by conversion to anti-phase coherence and transfer to (1)H for detection. We observe substantial enhancements, up to ≫200-fold, relative to the dark (laser off) experiment. Resonances of both side-chain and backbone CH pairs are enhanced for the three aromatic residues Trp, His, and Tyr, the σ(32) peptide, and the drkN SH3 protein. The sensitivity of this experiment, defined as signal-to-noise per square root of unit time (S/N)(t), is unprecedented in the NMR polarization enhancement literature dealing with polypeptides in solution. Up to a 16-fold larger (S/N)(t) than for the (1)H-(13)C SE-HSQC reference sequence is achieved, for the σ(32) peptide. Data collection time is reduced up to 256-fold, highlighting the advantages of (1)H-detected (13)C photo-CIDNP in solution NMR.

Abstract 2993: Homologous recombination between telomeres is present in ALT and telomerase-positive immortal cells

Abstract Telomere homeostasis is a hallmark of malignancy and occurs via two different mechanisms. The first mechanism is telomere length control via telomerase activity, the second mechanism is characterized by the lack of any detectable telomerase activity and is known as the alternative lengthening of telomeres (ALT) pathway. There is good evidence that homologous recombination between telomeric DNA is responsible for telomere elongation in these cells and therefore enables infinite growth. Telomeres resemble fragile DNA sites, and their repetitive sequence poses a challenge for the DNA replication machinery. Replication fork stallments have been shown to occur in mammalian telomeres, giving rise to DNA lesions and genetic instability. DNA replication can resume via a homologous recombination process, which could potentially cause telomere elongation. Although telomerase-positive human cells are believed to not recombine their telomeres, we were challenged by the question whether telomeric recombination is a rare event that is missed with an insensitive detection technique. In order to address this issue we designed a novel, sensitive and specific recombination reporter system that demonstrates recombination events that have become detectable by gaining antibiotic resistance. For this purpose cells were first tagged with a subtelomeric and subsequently with a telomeric reporter cassette, after which individual clones were obtained. Once telomeres recombined, both cassettes co-localize on the same telomere. Alternative splicing of intervening sequences between two portions of a resistance gene results in antibiotic resistance, hence allowing the specific and sensitive detection of a telomeric recombination event occurring upstream of the telomeric tag. With this reporter system we show that telomeric recombination is present in ALT and telomerase-positive human fibroblasts. The frequency of recombination does not differ between these two cell lines. DNA damage caused by UV irradiation, change in heterochromatin formation from HDAC inhibition or by demethylating DNA with azacitidine did not change the frequency of homologous recombination in either ALT or telomerase-positive clones. In contrast, treatment of cells with aphidicolin, which renders telomeres fragile, significantly increased the frequency of telomeric recombination in ALT, but not in telomerase-positive cells. The introduction of telomerase into these ALT cell clones abolished the effect of the aphidicolin-induced homologous recombination. Our data show that in telomerase-positive cells and ALT cells homologous recombination between telomeres occurs with nearly the same frequency, but that the increase in telomeric recombination via fragile telomeres is dependent on the absence of telomerase activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2993. doi:10.1158/1538-7445.AM2011-2993

PIXE analysis of multilayer targets

AbstractParticle induced X‐ray emission (PIXE) analysis is generally accepted to be a depth insensitive technique and most of the work up until recently has been oriented to the analysis of in‐depth homogeneous samples. Up until recently, multilayer targets were normally looked upon as a source of problems, implying a limitation to qualitative or semi‐quantitative approaches. The growing need to analyse layered targets having complex structures or close Z elements, which provide unsolvable Rutherford backscattering spectrometry (RBS) spectra, pointed out the need to change this situation. The coupling of two software codes, namely NDF and DATTPIXE, was thus carried out a few years ago based on the development of a PIXE yields simulation library, LibCPIXE. Since then, developments were introduced at various levels, from the software code level to the level of analytical methodologies and even equipment. Starting in the concepts of equivalent depth and differential PIXE, and ending in the possibilities opened up by the use of microcalorimeter high‐resolution detectors within this context, an overview of the current status of development of the analytical technology involved is made. Copyright © 2011 John Wiley & Sons, Ltd.

Pseudo-continuous transfer insensitive labeling technique

Transfer insensitive labeling technique (TILT) was previously applied to acquire multislice cerebral blood flow maps as a pulsed arterial spin labeling (PASL) method. The magnetization transfer effect with TILT is well controlled by using concatenated radiofrequency pulses. However, use of TILT has been limited by several challenges, including slice profile errors, sensitivity to arterial transit time and intrinsic low signal-to-noise ratio (SNR). In this work, we propose to address these challenges by making the original TILT method into a novel pseudo-continuous arterial spin labeling approach, named pseudo-continuous transfer insensitive labeling technique (pTILT). pTILT improves perfusion acquisitions by (i) realizing pseudo-continuous tagging with nonadiabatic pulses, (ii) being sensitive to slow flows in addition to fast flows, and (iii) providing flexible labeling geometries. Perfusion maps during both resting state and functional tasks are successfully demonstrated in healthy volunteers with pTILT. A comparison with typical SNR values from other perfusion techniques shows that although pTILT provides less SNR than inversion-based pseudo-continuous ASL techniques, the modified sequence provides similar SNR to inversion-based PASL techniques.

Quantitative immunofluorescent blotting of the Multidrug Resistance-associated Protein 2 (MRP2)

Introduction Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. Purpose The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-Binding Cassette Transporter Isoform C2/Multidrug Resistance-associated Protein 2 (ABCC2/MRP2). Methods Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotted with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. Results The limits of quantitation for the time- insensitive technique described here were from 0.001 μg to 0.5 μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r 2 values were 0.986 ± 0.012. The utility and sensitivity of this technique were demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. Discussion The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings.

Detection of a Substantial Rate of Multidrug‐Resistant Tuberculosis in an HIV‐Infected Population in South Africa by Active Monitoring of Sputum Samples

Tuberculosis (TB) coinfection with human immunodeficiency virus (HIV) is a substantial problem in South Africa. There has been a presumption that drug-resistant strains of TB are common in South Africa, but few studies have documented this impression. In Phidisa, a joint observational and randomized HIV treatment study for South African National Defence Force members and dependents, an initiative was launched to test subjects (by use of microbiologic TB test) who appeared to be at high risk. We report results for HIV-infected subjects. TB was identified by culture in 116 (19.9%) of 584 patients selected for sputum examination on the basis of suggestive symptoms. Smear was an insensitive technique for confirming the diagnosis: only 33% of culture-positive patients were identified by smear, with a 0.2% false-positive rate. Of the 107 culture-positive individuals with susceptibility testing, 22 (20.6%) were identified to be multidrug resistant (MDR), and 4 (3.7%) were identified to be extensively drug resistant. Culture-positive cases with a history of TB treatment had more than twice the rate of MDR than those without (27.1% vs 11.9%; P = .05). TB is common in this cohort of HIV-infected patients. Smear was not a sensitive technique for identifying culture-positive cases in this health system. Drug susceptibility testing is essential to proper patient management because MDR was present in 20.6% of culture-positive patients. Better management strategies are needed to reduce the development of MDR TB, because so many of these patients had received prior antituberculous therapy that was presumably not curative.

AIT test has no problem in the detection of anti-ribosomal P – authors' response

In a recent article, Jearn and Kim assessed the ability of indirect immunofluorescence on a novel human macrophage cell line (IT-1) in an autoimmune target (AIT) assay to predict the presence of anti-ribosomal P (anti-Rib-P) antibodies [1], based on a previously described cytoplasmic and/or nucleolar staining pattern [2]. Despite the clearance of the AIT test by the South Korea Food and Drug Administration, to the best of our knowledge this test is not widely used as a screening method – neither is it approved by the Food and Drug Administration (FDA) USA or is it a Communaute Europeenne (CE) certified method for the detection of antinuclear antibodies. By contrast, many laboratories rely on HEp-2 cell substrates that are widely available as FDA and CE approved diagnostic kits from a variety of manufacturers. In their study, Jearn and Kim identified sera with anti-Rib-P reactivity based on a double immunodiffusion (ID) assay, and then used these sera to determine the positivity and staining patterns in the AIT test. This method raises concerns because it is generally known that immunodiffusion is a relatively insensitive technique for the detection of human autoantibodies when compared with other techniques such as ELISA and western immunoblotting (WB). In an example relevant to the anti-Rib-P system, Bonfa and colleagues clearly demonstrated that immunodiffusion has very low sensitivity for the detection of anti-Rib-P antibodies [3]. In their study, only 14% of anti-Rib-P-positive samples identified by WB were detected by immunodiffusion. Another study showed that ID exhibits significantly lower sensitivity compared with a multiplex system: 63/130 (48.5%) samples with at least one positive extractable nuclear antibody result were negative by ID, and all 11 sera with anti-Rib-P reactivity did not show a typical cytoplasmic staining pattern by indirect immunofluorescence [4]. While the aforementioned studies used HEp-2 cells as the substrate, there would be no reason to believe that the AIT assay would perform remarkably better. In the context of contemporary diagnostic assays such as WB, ELISA or multiplexed addressable laser bead immuno-assays, therefore, the data presented by Jearn and Kim do not support the claim that the AIT test represents a sensitive and reliable method for the detection of anti-Rib-P antibodies. A systematic study is mandatory to compare the sensitivity and specificity of the AIT test for the detection of anti-Rib-P antibodies and other antibody specificities using a variety of contemporary diagnostic platforms, not just ID. In addition to anti-Rib-P, such a study could also include the evaluation of anti-Jo-1, anti-SS-A (Ro60) and anti-Ro52 antibodies, which have been shown to produce false negative results in indirect immunofluorescence on HEp-2 cells [2,5].

Quantitative evaluation of sebum lipid components with nuclear magnetic resonance

A NMR spectroscopic method is described that enables the quantitation of specific lipid classes and components, independent of fatty acid composition. We demonstrate this method for measuring cholesterol, squalene, and pools of sterol esters, wax esters (WEs), and triglyceride (TG) components in sebum and meibum. When 600 MHz NMR equipment is used in conjunction with highly sensitive cryogenically cooled probes, this method has adequate sensitivity, and for some applications, advantages over commonly used HPLC-evaporative light-scattering detection and mass spectrometry-based approaches. This method is shown to be useful for preclinical and clinical monitoring of the efficacy of sebum-reducing agents in animals and humans. In Syrian hamsters, 3% topical flutamide and 20 mg/kg oral isotretinoin reduced sterol esters by 18.7% and 30.0%, respectively, and reduced WEs by 32.9% and 31.8%, respectively, as measured in a punch biopsy of the ear. In a 72 patient clinical methodology study, the assay delivered reproducible and noninvasive measurements of WEs, cholesteryl esters, TGs, and squalene from Sebutape skin blots. The quantitative results of sebum analysis obtained by the NMR method correlate well with those obtained with HPLC-based approaches. This approach may be broadly applicable to cases in which fatty acid-independent quantification of lipid classes is desired.

Identification of alphaB-crystallin, a biomarker of renal cell carcinoma by SELDI-TOF MS

Spectrometric-based surface-enhanced laser desorption/ionization ProteinChip (SELDI-TOF) facilitates rapid and easy analysis of protein mixtures and is often exploited to define potential diagnostic markers from sera. However, SELDI- TOF is a relatively insensitive technique and unable to detect circulating proteins at low levels even if they are differentially expressed in cancer patients. Therefore, we applied this technology to study tissues from renal cell carcinomas (RCC) in comparison to healthy controls. We found that different biomarkers are identified from tissues than those previously identified in serum, and that serum markers are often not produced by the tumors themselves at detectable levels, reflecting the nonspecific nature of many circulating biomarkers. We detected and characterized áB-crystallin as an overexpressed protein in RCC tissues and showed differential expression by immunohistochemistry. We conclude that SELDI-TOF is more useful for the identification of biomarkers that are synthesized by diseased tissues than for the identification of serum biomarkers and identifies a separate set of markers. We suggest that SELDI-TOF should be used to screen human cancer tissues to identify potential tissue-specific proteins and simpler and more sensitive techniques can then be applied to determine their validity as biomarkers in biological fluids.

Identification of αB-Crystallin, a Biomarker of Renal Cell Carcinoma by SELDI-TOF MS

Spectrometric-based surface-enhanced laser desorption/ionization ProteinChip (SELDI-TOF) facilitates rapid and easy analysis of protein mixtures and is often exploited to define potential diagnostic markers from sera. However, SELDI- TOF is a relatively insensitive technique and unable to detect circulating proteins at low levels even if they are differentially expressed in cancer patients. Therefore, we applied this technology to study tissues from renal cell carcinomas (RCC) in comparison to healthy controls. We found that different biomarkers are identified from tissues than those previously identified in serum, and that serum markers are often not produced by the tumors themselves at detectable levels, reflecting the nonspecific nature of many circulating biomarkers. We detected and characterized áB-crystallin as an overexpressed protein in RCC tissues and showed differential expression by immunohistochemistry. We conclude that SELDI-TOF is more useful for the identification of biomarkers that are synthesized by diseased tissues than for the identification of serum biomarkers and identifies a separate set of markers. We suggest that SELDI-TOF should be used to screen human cancer tissues to identify potential tissue-specific proteins and simpler and more sensitive techniques can then be applied to determine their validity as biomarkers in biological fluids.