Diagnostic Performance of a Lower-dose Contrast-Enhanced 4D Dynamic MR Angiography of the Lower Extremities at 3 T Using Multisegmental Time-Resolved Maximum Intensity Projections.

BACKGROUND: For peripheral artery disease (PAD), MR angiography (MRA) is a well-established diagnostic modality providing morphologic and dynamic information comparable to digital subtraction angiography (DSA). However, relatively large amounts of contrast agents are necessary to achieve this. PURPOSE: To evaluate the diagnostic accuracy of time-resolved 4D MR-angiography with interleaved stochastic trajectories (TWIST-MRA) by using maximum intensity projections (MIPs) of dynamic images acquired with reduced doses of contrast agent. STUDY TYPE: Retrospective. POPULATION: Forty adult PAD patients yielding 1088 artery segments. FIELD STRENGTH/SEQUENCE: A 3.0 T, time-resolved 4D MR-angiography with TWIST-MRA and MIP of dynamic images. ASSESSMENT: DSA was available in 14 patients (256 artery segments) and used as reference standard. Three-segmental MIP reconstructions of TWIST-images after administration of 3 mL of gadolinium-based contrast agent (Gadoteridol/Prohance®, 0.5 M) per anatomical level (pelvis, thighs, and lower legs) yielded 256 artery segments for correlation between MRA and DSA. Three independent observers rated image quality (scale: 1 [nondiagnostic] to 4 [excellent]) and the degree of venous overlay (scale: 0 [none] to 2 [significant]) for all segments. Diagnostic accuracy for the detection of >50% stenosis and artery occlusion was calculated for all observers. STATISTICAL TESTS: Binary classification test (sensitivity, specificity, positive/negative predictive values, diagnostic accuracy). Intraclass correlation coefficients (ICCs), logistic regression analysis with comparison of areas under the receiver-operating-characteristics (ROC) curves (AUCs) with the DeLong method. Bland-Altman-comparison. RESULTS: High diagnostic performance was achieved for the detection of >50% stenosis (sensitivity 92.9% [84.3-99.9% (95%-CI)] and specificity 98.5% [95.7-99.8% (95%-CI)]) and artery occlusion (sensitivity 93.1% [77.2-99.2% (95%-CI)] and specificity 99.1% [96.9-99.9% (95%-CI)]). Inter-reader agreement was excellent with ICC values ranging from 0.95 to 1.0 for >50% artery stenosis and occlusion. Image quality was good to excellent for both readers (3.41 ± 0.72, 3.33 ± 0.65, and 3.38 ± 0.61 [mean ± SD]) with good correlation between observer ratings (ICC 0.71-0.81). No significant venous overlay was observed (0.06 ± 0.24, 0.23 ± 0.43 and 0.11 ± 0.45 [mean ± SD]). DATA CONCLUSION: MIPs of dynamic TWIST-MRA offer a promising diagnostic alternative necessitating only reduced amounts (50%) of gadolinium-based contrast agents for the entire runoff vasculature. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 2.

In-Tip Lanthanum Oxide Monolith for the Enrichment of Phosphorylated Biomolecules.

Polymeric monoliths fabricated in tips with embedded materials of choice are important in separation science. Polymeric backbone however interferes in the enrichment and thus affects efficiency. This work focuses on the in-tip fabrication of lanthanum oxide porous monolith and its application in the enrichment of phosphorylated peptides and lipids. Polycondensation reaction uses an aqueous solution of LaCl3·7H2O with N-methyl formamide as porogen and propylene oxide as initiator. The aging time of monolith and temperature condition for the reaction are optimized to attain porous monolithic tip. A comparison of (i) solid phase batch extraction using La2O3, (ii) La2O3 embedded in poly(glycidyl methacrylate (GMA)/divinylbenzene (DVB)) tip, and (iii) pure La2O3 monolithic tip shows improved enrichment efficiency in the case of pure La2O3 monolithic tip. The monolithic tip achieves selectivity of 1:4500 as compared to solid phase extraction (SPE)(1:3500) and limit of detection down to 0.25 fmol. The in-tip La2O3 monolith strategy has better batch to batch reproducibility, reduced time of enrichment, and ease of operation in comparison to solid phase batch extraction. The developed strategy enriches phospho- content from biological samples like phosvitin and lipovitellin from egg yolk and phospholipids/phosphopeptides from human serum. The enriched phospho- moieties are analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) except the phospholipids where laser desorption ionization (LDI)-MS is employed.

Glass-Fiber-based MR-safe Guidewire for MR Imaging-guided Endovascular Interventions: In Vitro and Preclinical in Vivo Feasibility Study.

Purpose To evaluate glass-fiber-based guidewires that are safe for magnetic resonance (MR) imaging-guided endovascular interventions by using a phantom and an in vivo swine model. Materials and Methods MR imaging-safe guidewires were made from micropultruded glass and/or aramid fibers and epoxy resin with diameters of 0.89 mm (0.035 inch) for standard and stiff guidewires and 0.36 mm (0.014 inch) for micro guidewires. MR imaging visibility and mechanical properties were assessed in a pulsatile flow model. After approval was obtained from the institutional animal care and use committee, MR imaging guidewires were evaluated for standard endovascular procedures in nine pigs. Real-time steady-state free-precession sequences were used for MR imaging-guided catheterization, balloon dilation, and stent implantation into aorto-iliac/visceral arteries and the vena cava (temporal resolution, five images per second; and spatial resolution, 150-mm field of view, and 128 × 128 matrix) with a 1.5-T clinical imager. Visualization with the guidewires was rated on a four-point scale, handling was rated on a three-point scale, and catheterization times for different vessel regions were determined by two interventional radiologists. Afterward, handling ratings and catheterization times were obtained for standard nitinol guidewires during x-ray-based fluoroscopy. Cannulation times, signal intensity in each vessel region, and visualization and handling ratings were measured for the MR imaging guidewires. Bland-Altman analysis was performed for inter- and intraobserver variability of cannulation time. Spearman rank correlation was used to compare handling of MR imaging guidewires and standard nitinol guidewires. Results MR imaging guidewires were characterized by good to excellent visibility, with a continuous artifact of 2 mm in diameter and 4 × 8-mm ball-shaped tip marker. Stiffness, flexibility, and guidance reflected comparable times for all in vitro and in vivo procedures with both the MR imaging and standard nitinol guidewires. Standard and micro MR imaging guidewires were most suitable for the iliac crossover maneuver. Phantom visceral artery cannulation was easier with standard and micro MR imaging guidewires. The stiff MR imaging guidewire provided the best support for cannulation of the swine aorta and vena cava. All interventional procedures were performed successfully without complications. Conclusion Preliminary results showed that the use of glass-fiber-based guidewires for evaluation of MR imaging-guided endovascular interventions is technically feasible and safe in a swine model, and potentially, in humans. © RSNA, 2017 Online supplemental material is available for this article.

Gadolinium oxide: Exclusive selectivity and sensitivity in the enrichment of phosphorylated biomolecules.

Selectivity and sensitivity define the dynamic applicability of separation and enrichment techniques. Owing to proteome complexity, numbers of separation media have been introduced in phosphoproteomics. Complex samples are pretreated to make the low-abundance molecules detectable by mass spectrometry. Gadolinium oxide nanoparticles, offering mono- and bi-dentate interactions, are optimized to capture the phosphopeptides. Selectivity of 1:11 000 is achieved for digested ß-casein phosphopeptides in bovine serum albumin digest background using gadolinium oxide nanoparticles. The limit of detection goes down to 1 attomole. With the optimized sample preparation protocol, gadolinium oxide nanoparticles enrich phosphopeptides of κ-casein (Ser148 and Ser170 ) from digested milk sample, fibrinogen alpha chain phosphopeptide (Ser609 ) along with four hydrolytic products of Ser22 -modified phosphopeptides from serum.

Newly fabricated magnetic lanthanide oxides core-shell nanoparticles in phosphoproteomics.

Metal oxides show high selectivity and sensitivity toward mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic core-shell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR, and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, nonfat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no nonspecific bindings. The selectivity is achieved up to 1:8500 using ß-casein/BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the reuse of core-shell nanoparticles up to four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential toward prostate cancer. Thus, the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics.

Body mass index is not a clinically meaningful predictor of patient reported outcomes of primary hip replacement surgery: prospective cohort study.

OBJECTIVES: To describe whether body mass index (BMI) is a clinically meaningful predictor of patient reported outcomes following primary total hip replacement (THR) surgery. DESIGN: Combined data from prospective cohort studies. We obtained information from four cohorts of patients receiving primary THR for osteoarthritis: Exeter Primary Outcomes Study (EPOS) (n = 1431); EUROHIP (n = 1327); Elective Orthopaedic Centre (n = 2832); and St. Helier (n = 787). The exposure of interest was pre-operative BMI. Confounding variables included: age, sex, SF-36 mental health, comorbidities, fixed flexion, analgesic use, college education, OA in other joints, expectation of less pain, radiographic K&L grade, ASA grade, years of hip pain. The primary outcome was the Oxford Hip Score (OHS). Regression models describe the association of BMI on outcome adjusting for all confounders. RESULTS: For a 5-unit increase in BMI, the attained 12-month OHS decreases by 0.78 points 95%CI (0.27-1.28), P-value 0.001. Compared to people of normal BMI (20-25), those in the obese class II (BMI 35-40) would have a 12-month OHS that is 2.34 points lower. Although statistically significant this effect is small and not clinically meaningful in contrast to the substantial change in OHS seen across all BMI groupings. In obese class II patients achieved a 22.2 point change in OHS following surgery. CONCLUSIONS: Patients achieved substantial change in OHS after THR across all BMI categories, which greatly outweighs the small difference in attained post-operative score. The findings suggest BMI should not present a barrier to access THR in terms of PROMs.

Preparation and evaluation of monolithic poly(N-vinylcarbazole-co-1,4-divinylbenzene) capillary columns for the separation of small molecules.

Short-term polymerization or the so-called low-conversion polymerization was applied for the preparation of N-vinylcarbazole (NVC) and 1,4-divinylbenzene (DVB) monolithic capillary columns. The synthesis was carried out by thermally initiated free radical copolymerization under the influence of inert micro- (toluene) and macroporogen (1-decanol) and α,α'-azoisobutyronitrile (AIBN) as radical initiator. The morphological and porous properties were studied by scanning electron microscopy (SEM), nitrogen adsorption, and mercury intrusion porosimetry (MIP). The copolymerization process was studied by monomer conversion measurements. This approach led to increased porosity and specific surface area. A specific surface area above 400 m(2)/g of the monolith and a distinct bimodal pore size distribution were obtained. The chromatographic performance was determined in terms of theoretical plate heights and number of theoretical plates. The lowest plate height value was found to be 3.9 µm (corresponding to ≈256,000 plates per meter) applying methylparaben utilizing an 80 mm × 0.2 mm i.d. monolithic capillary. The developed NVC/DVB monolithic supports showed high separation efficiency towards small molecules, which was exemplified applying reversed-phase (RP) separation of alkylbenzenes, beta-blockers, flavanoids, parabens, and phenones. The loading capacity was analyzed for isocratic separation of seven alkylbenzenes and was found to be up to 77 ng total mass of alkylbenzenes. Furthermore, a long-term stability test of 1,000 consecutive runs was performed and resulted in a maximum variance of 0.97, 0.85, and 0.16 % RSD for resolution, peak width at half height, and retention times, respectively. The material was proven to have a high permeability of 1.11E-14 m(2), applying water as a mobile phase.

Au-nanomaterials as a superior choice for near-infrared photothermal therapy.

Photothermal therapy (PPT) is a platform to fight cancer by using multiplexed interactive plasmonic nanomaterials as probes in combination with the excellent therapeutic performance of near-infrared (NIR) light. With recent rapid developments in optics and nanotechnology, plasmonic materials have potential in cancer diagnosis and treatment, but there are some concerns regarding their clinical use. The primary concerns include the design of plasmonic nanomaterials which are taken up by the tissues, perform their function and then clear out from the body. Gold nanoparticles (Au NPs) can be developed in different morphologies and functionalized to assist the photothermal therapy in a way that they have clinical value. This review outlines the diverse Au morphologies, their distinctive characteristics, concerns and limitations to provide an idea of the requirements in the field of NIR-based therapeutics.

Monolithic poly(N-vinylcarbazole-co-1,4-divinylbenzene) capillary columns for the separation of biomolecules.

Monolithic capillary columns were prepared by thermally initiated free radical copolymerization of N-vinylcarbazole (NVC) and 1,4-divinylbenzene (DVB) within the confines of 200 and 100 µm i.d. fused silica capillaries. The reaction was carried out under the influence of inert micro-(toluene) and macroporogen (1-decanol) and α,α'-azoisobutyronitrile (AIBN) as a free radical initiator. The material proved high mechanical stability applying water and acetonitrile as mobile phases. The morphological and porous properties were studied by scanning electron microscopy (SEM), nitrogen sorption (BET) and mercury intrusion porosimetry (MIP). The homogeneity of the copolymerization process was confirmed by elemental analysis and monomer conversion measurements. The newly developed NVC/DVB monolithic supports showed high separation efficiency towards biomolecules, applying reversed-phase (RP) and ion-pair reversed-phase (IP-RP) separation modes, which is exemplified by the separations of peptides, proteins and oligonucleotides. Furthermore the maximum loading capacity was evaluated. The chromatographic performance under isocratic elution was determined in terms of theoretical plate number and plate height, where up to 41,000 plates per column and a minimum plate height value of 1.7 µm were achieved, applying oligonucleotide separations. In gradient elution mode, peak capacities of 96 and 127 were achieved within a gradient time window of 60 min for protein and oligonucleotide separations, respectively. The material proved to have high permeability, good repeatability of the fabrication process and high surface areas in the range of 120-160 m(2) g(-1).

Noninferiority of Monoparametric MRI Versus Multiparametric MRI for the Detection of Prostate Cancer: Diagnostic Accuracy of ADC Ratios Based on Advanced "Zoomed" Diffusion-Weighted Imaging.

OBJECTIVES: The aim of this study was to compare the diagnostic accuracy of apparent diffusion coefficient (ADC) ratios as a monoparametric magnetic resonance imaging (MRI) protocol for the detection of prostate cancer (PCa) with the established multiparametric (mp) MRI at 3.0 T. MATERIALS AND METHODS: According to power analysis, 52 male patients were included in this monocenter study with prospective data collection and retrospective, blinded multireader image analysis. The study was approved by the local ethics committee. Patients were recruited from January to December 2020. Based on mpMRI findings, patients underwent in-bore MR biopsy or prostatectomy for histopathologic correlation of suspicious lesions. Three readers, blinded to the histopathologic results and images of mpMRI, independently evaluated ADC maps for the detection of PCa. The ADC ratio was defined as the lowest signal intensity (SI) of lesions divided by the SI of normal tissue in the zone of origin. Predictive accuracy of multiparametric and monoparametric MRI were compared using logistic regression analysis. Moreover, both protocols were compared applying goodness-of-fit analysis with the Hosmer-Lemeshow test for continuous ADC ratios and Pearson χ2 test for binary decision calls, correlation analysis with Spearman ρ and intraclass correlation coefficients, as well as noninferiority assessment with a TOST ("two one-sided test"). RESULTS: Eighty-one histopathologically proven, unique PCa lesions (Gleason score [GS] ≥ 3 + 3) in 52 patients could be unequivocally correlated, with 57 clinically significant (cs) PCa lesions (GS ≥ 3 + 4). Multiparametric MRI detected 95%, and monoparametric ADC detected ratios 91% to 93% of csPCa. Noninferiority of monoparametric MRI was confirmed by TOST (P < 0.05 for all comparisons). Logistic regression analysis revealed comparable predictive diagnostic accuracy of ADC ratios (73.7%-87.8%) versus mpMRI (72.2%-84.7%). Spearman rank correlation coefficient for PCa aggressiveness revealed satisfactory correlation of ADC ratios (P < 0.013 for all correlations). The Hosmer-Lemeshow test for the logistic regression analysis for continuous ADC ratios indicated adequate predictive accuracy (P = 0.55-0.87), and the Pearson χ2 test showed satisfactory goodness of fit (P = 0.35-0.69, χ2 = 0.16-0.87). CONCLUSIONS: Normalized ADC ratios based on advanced DWI are noninferior to mpMRI at 3.0 T for the detection of csPCa in a preselected patient cohort and proved a fast and accurate assessment tool, thus showing a potential prospect of easing the development of future screening methods for PCa.

Comprehensive proteomic and transcriptomic characterization of hepatic expression signatures affected in p14 liver conditional knockout mice.

Scaffold proteins regulate intracellular MAP kinase signaling by providing critical spatial and temporal specificities. We have shown previously that the scaffold protein MEK1 partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in livers of mice (p14(Δhep) ) we analyzed protein and transcript signatures in tissue samples. Further biological network analysis predicted that the differentially expressed transcripts and proteins are involved in cell cycle progression and regulation of cellular proliferation. Although some of the here identified signatures were previously linked to phospho-ERK activity, most of them were novel targets of the late endosomal p14/MP1/MEK/ERK signaling module. Finally, the proliferation defect was confirmed in a chemically induced liver regeneration model in p14(Δhep) knockout mice.

Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS.

INTRODUCTION: Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. OBJECTIVE: This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. METHODOLOGY: The specific focus of the study is on thin-layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf-MELDI-MS). Samples employed in the study were standards and microwave-assisted water extracts from Quercus. RESULTS: TLC analysis proved the presence of mono-, di- and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC-MS confirmed data obtained via TLC for mono- to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf-MELDI-MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf-MELDI-MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. CONCLUSION: Evaluation of all three techniques employed, clearly proved the heightened performance of mf-MELDI-MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC-MS is suitable for quantitative analysis.

Proteomic analysis of endosomes from genetically modified p14/MP1 mouse embryonic fibroblasts.

The p14/MP1 scaffold complex binds MEK1 and ERK1/2 on late endosomes, thus regulating the strength, duration and intracellular location of MAPK signaling. By organelle proteomics we have compared the protein composition of endosomes purified from genetically modified p14⁻/⁻, p14+/⁻ and p14(rev) mouse embryonic fibroblasts. The latter ones were reconstituted retrovirally from p14⁻/⁻ mouse embryonic fibroblasts by reexpression of pEGFP-p14 at equimolar ratios with its physiological binding partner MP1, as shown here by absolute quantification of MP1 and p14 proteins on endosomes by quantitative MS using the Equimolarity through Equalizer Peptide strategy. A combination of subcellular fractionation, 2-D DIGE and MALDI-TOF/TOF MS revealed 31 proteins differentially regulated in p14⁻/⁻ organelles, which were rescued by reexpression of pEGFP-p14 in p14⁻/⁻ endosomes. Regulated proteins are known to be involved in actin remodeling, endosomal signal transduction and trafficking. Identified proteins and their in silico interaction networks suggested that endosomal signaling might regulate such major cellular functions such as proliferation, differentiation, migration and survival.

Online coupling of thin layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: synthesis and application of a new material for the identification of carbohydrates by thin layer chromatography/matrix free material enhanced laser desorption/ionization mass spectrometry.

This article describes the online hyphenation of thin layer chromatography with matrix free material enhanced laser desorption/ionization mass spectrometry (mf-MELDI-MS), the preparation of new material for MELDI and application of this newly synthesized material using TLC/MELDI-MS for the analysis of carbohydrate reference standards and plant extracts. Samples included within these analyses are standard solutions of glucose, sucrose, raffinose and a plant extract of Quercus robur, which is used for its anti-inflammatory, anti-viral and anthelminitc properties in phytomedicine. A new material for mf-MELDI-MS is prepared by immobilizing bradykinin--a peptide, on silica gel coupled to 4-(3-triethoxysilylpropylureido)azobenzene. This modification enables the absorption of laser energy sufficient for desorption and ionization of low molecular weight molecules like carbohydrates and amino acids. The newly synthesized material delivered excellent results in respect to signal-to-noise (S/N) ratio (S/N ratio: >9/1) and sensitivity (limit of detection (LOD): lower to ng/microL). Hyphenation of TLC to MELDI-MS employing the novel developed material simultaneously as chromatographic and mass spectrometric sorbent was shown for the first time for the analysis of low molecular weight molecules like mono- and oligosaccharides.

Mechanical failure of 113 uncemented modular revision femoral components.

AIMS: We evaluated a large database with mechanical failure of a single uncemented modular femoral component, used in revision hip arthroplasty, as the end point and compared them to a control group treated with the same implant. Patient- and implant-specific risk factors for implant failure were analyzed. METHODS: All cases of a fractured uncemented modular revision femoral component from one manufacturer until April 2017 were identified and the total number of implants sold until April 2017 was used to calculate the fracture rate. The manufacturer provided data on patient demographics, time to failure, and implant details for all notified fractured devices. Patient- and implant-specific risk factors were evaluated using a logistic regression model with multiple imputations and compared to data from a previously published reference group, where no fractures had been observed. The results of a retrieval analysis of the fractured implants, performed by the manufacturer, were available for evaluation. RESULTS: There were 113 recorded cases with fracture at the modular junction, resulting in a calculated fracture rate of 0.30% (113/37,600). The fracture rate of the implant without signs of improper use was 0.11% (41/37,600). In 79% (89/113) of cases with a failed implant, either a lateralized (high offset) neck segment, an extralong head, or the combination of both were used. Logistic regression analysis revealed male sex, high body mass index (BMI), straight component design, and small neck segments were significant risk factors for failure. Investigation of the implants (76/113) showed at least one sign of improper use in 72 cases. CONCLUSION: Implant failure at the modular junction is associated with patient- and implant-specific risk factors as well as technical errors during implantation. Whenever possible, the use of short and lateralized neck segments should be avoided with this revision system. Implantation instructions and contraindications need to be adhered to and respected. Cite this article: Bone Joint J 2020;102-B(5):573-579.

Fluorescent isotope-coded affinity tag 2: peptide labeling and affinity capture.

Fluorescent isotope-coded affinity tag (FCAT) is a novel reagent to label cysteine-containing peptides. The fluorescein group on the tag enables absolute quantification by fluorescence detection, also supporting affinity capture of the labeled peptides. In this paper we report the synthesis of the heavy isotopic form of the FCAT reagent and its use in labeling tryptic peptides. The heavy form of the reagent exhibited the same reactivity and chromatographic behavior that of the light version. Effective labeling of tryptic peptides from alpha-lactalbumin, fetuin, BSA and phosphorylase b was attained by using both the heavy and light FCAT tags. Selective capture of the FCAT-labeled peptides was easily performed by pipette tips containing either anti-FITC antibody or iminodiacetic-acid-coated beads. The differently labeled peptides were separated by RP HPLC and analyzed by MALDI-TOF MS.

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells.

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

Sample preparation for the analysis of complex carbohydrates by multicapillary gel electrophoresis with light-emitting diode induced fluorescence detection.

This paper evaluates various sample preparation methods for multicapillary gel electrophoresis based glycan analysis to support electrokinetic injection. First the removal of excess derivatization reagent is discussed. Although the Sephadex G10 filled multiscreen 96-well filter plate and Sephadex G10 filled pipet tips enabled increased analysis sensitivity, polyamide DPA-6S pipet tips worked particularly well. In this latter case an automated liquid handling system was used to increase purification throughput, necessary to feed the multicapillary electrophoresis unit. Problems associated with the high glucose content of such biological samples as normal human plasma were solved by applying ultrafiltration. Finally, a volatile buffer system was developed for exoglycosidase-based carbohydrate analysis.

Novel monolithic poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) capillary columns for biopolymer separation.

Hydrophobic organo-silane based monolithic capillary columns were prepared by thermally initiated free radical polymerisation within the confines of 200 microm i.d. fused silica capillaries. A novel crosslinker, namely bis(p-vinylbenzyl)dimethylsilane (BVBDMS), was copolymerised with p-methylstyrene (MS) in the presence of 2-propanol and toluene, using alpha,alpha'-azoisobutyronitrile (AIBN) as initiator. Monolithic capillary columns, differing in the total monomer, microporogen content and microporogen nature were fabricated and the chromatographic efficiency of each monolith, regarding the separation of proteins, peptides and oligonucleotides, was evaluated and compared. Changes in monolith morphology were monitored by scanning electron microscopy (SEM). Porosity and specific surface areas of the supports were studied by means of mercury intrusion porosimetry and BET measurements, respectively. Pressure drop vs. flow rate measurements proved the prepared poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) (MS/BVBDMS) monoliths to be mechanically stable and swelling propensity (SP) factors of 0.78-1.10 indicate high crosslinking homogeneity.