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A number of lipoprotein(a) [Lp(a)] analytical techniques are available that quantify distinct particle components, yet their clinical efficacy has not been comprehensively evaluated. This study determined whether Lp(a) mass [Lp(a)-M], Lp(a) cholesterol content [Lp(a)-C], and particle concentration [Lp(a)-P] differentially discriminated risk of calcific aortic valve disease (CAVD) or incident coronary heart disease (CHD) among 4679 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Lp(a)-M, Lp(a)-C, and Lp(a)-P were measured in individuals without clinical evidence of CHD at baseline. Relative risk regression and Cox proportional analysis determined associations between Lp(a) and the presence of CAVD or 12-year risk of CHD, respectively. To control for the relatively high lower limits of quantification for Lp(a)-C and Lp(a)-P assays, the upper 25th and 15th percentiles were selected as analytical cutoff points. Regardless of method or analytical cutoff, high Lp(a) concentrations were significantly associated with CAVD and CHD in MESA participants following adjustment for typical cardiovascular risk factors. Stratifying by race/ethnicity rendered most associations nonsignificant after correction for multiple comparisons, but Lp(a) remained associated with CAVD in whites irrespective of method (all < 0.0001). Associations of Lp(a)-C, Lp(a)-P, and Lp(a)-M with CAVD or incident CHD were similar in this entire MESA sample using a dichotomized statistical approach. However, the high lower limits of quantification and imprecision of the Lp(a)-C and Lp(a)-P assays limited their usefulness in our analyses and would likely do so in research and clinical settings.

In this placebo-controlled, double-blind, crossover human feeding study, the effects of polydextrose (PDX; 8 g/d) on the colonic microbial composition, immune parameters, bowel habits and quality of life were investigated. PDX is a complex glucose oligomer used as a sugar replacer. The main goal of the present study was to identify the microbial groups affected by PDX fermentation in the colon. PDX was shown to significantly increase the known butyrate producer Ruminococcus intestinalis and bacteria of the Clostridium clusters I, II and IV. Of the other microbial groups investigated, decreases in the faecal Lactobacillus–Enterococcus group were demonstrated. Denaturing gel gradient electrophoresis analysis showed that bacterial profiles between PDX and placebo treatments were significantly different. PDX was shown to be slowly degraded in the colon, and the fermentation significantly reduced the genotoxicity of the faecal water. PDX also affected bowel habits of the subjects, as less abdominal discomfort was recorded and there was a trend for less hard and more formed stools during PDX consumption. Furthermore, reduced snacking was observed upon PDX consumption. This study demonstrated the impact of PDX on the colonic microbiota and showed some potential for reducing the risk factors that may be associated with colon cancer initiation.

The mitochondrial oxidative phosphorylation (OXPHOS) system plays a pivotal role in the cell’s energy conversion. The enzymes involved in OXPHOS are arranged in five protein-lipid complexes. The first four complexes (I–IV) form the mitochondrial respiratory chain, while Complex V is an F 1 F o -ATP synthase. Mutations in genes involved in the biosynthesis of the OXPHOS complexes are an important cause of metabolic diseases. Blue-native polyacrylamide gel electrophoresis (BN-PAGE), originally developed by Hermann Schägger in the 1990s, has become instrumental in gaining insights into structure/function relationships of the OXPHOS system, including: (1) the assembly pathways of the complexes, (2) the composition of higher-order respiratory chain supercomplexes and (3) pathologic mechanisms in patients with a monogenetic OXPHOS disorder. We have used BN-PAGE for >20 years and validate here our recently published step-by-step laboratory protocol. This protocol describes the manual casting of native mini-gels and sample preparation for the resolution of individual OXPHOS complexes or respiratory chain supercomplexes. In addition to BN-PAGE, we explain the closely related clear-native (CN)-PAGE and two-dimensional BN/denaturing-PAGE techniques. Downstream applications include western blot analysis and in-gel enzyme activity staining for Complexes I, II, IV and V. Limitations of the technique are the comparative insensitivity of in-gel Complex IV activity staining and the lack of in-gel Complex III activity staining. Compared to other published BN-PAGE protocols, our protocol contains a shortened sample extraction procedure, advises when to use BN-PAGE and when to use CN-PAGE, and suggests a simple enhancement step for in-gel Complex V activity staining that markedly improves sensitivity. Our protocol is adaptable and yields robust, semi-quantitative and reproducible results.

Detecting antibiotics in the milk supply chain is crucial to protect humans from allergic reactions, as well as preventing the build-up of antibiotic resistance. The dairy industry has controls in place at processing facilities, but controls on dairy farms are limited to manual devices. Errors in the use of these manual devices can result in severe financial harm to the farms. This illustrates an urgent need for automated methods of detecting antibiotics on a dairy farm, to prevent the shipment of milk containing antibiotics. This work introduces the microchip capillary electrophoresis dairy device, a low-cost system that utilizes microchip capillary electrophoresis as well as fluorescence spectroscopy for the detection of ciprofloxacin contained in milk. The microchip capillary electrophoresis dairy device is operated under antibiotic-absent conditions, with ciprofloxacin not present in a milk sample, and antibiotic-present conditions, with ciprofloxacin present in a milk sample. The response curve for the microchip capillary electrophoresis dairy device is found through experimental operation with varied concentrations of ciprofloxacin. The sensitivity and limit of detection are quantified for the microchip capillary electrophoresis dairy device.

Serum albumin measurement is an important parameter routinely evaluated in clinical biochemistry within the livestock industry. It plays a crucial role in assessing the nutritional and health status of animals, as well as in aiding the diagnosis of various pathological conditions as a complementary tool. Several laboratory methods are available for albumin measurement; however, some methods have been documented to overestimate the concentration of serum albumin versus the proposed gold standard of serum protein electrophoresis. The primary aim of the study was to analyze the agreement between albumin measurement in bovine serum samples by the capillary zone electrophoresis (CZE), agarose gel electrophoresis (AGE), bromocresol green (BCG), and purple (BCP) methods. In addition, AGE and CZE methods were also compared for quantitation of globulin fractions. Lastly, reference intervals were established using all methods using the American Society for Veterinary Clinical Pathology (ASVCP) guidelines. Serum samples from 55 clinically normal Brangus cattle (5 ± 1.5 years old) were examined by the four methods. For the albumin method comparison, all methods were significantly correlated ( r  = 0.55–0.91, p  < 0.0001) and the bias between methods ranged from 0.01–0.22 g/dL. For AGE and CZE methods, all protein fractions were significantly correlated ( r  = 0.85–0.91, p  < 0.0001) except for the alpha 1 fraction ( r  = 0.21, p  = 0.12). Five fractions were quantitated using AGE method: albumin, alpha 1, alpha 2, beta, and gamma globulins. For the CZE method, six fractions were resolved with the sub fractionation of beta 1 and beta 2 globulin fractions. The evaluated methods showed good agreement in determining albumin levels in cattle. Although CZE offers higher resolution, it requires careful interpretation and precise definition of fractions. Therefore, methodological choice and consideration of analyzer-specific reference intervals are essential for accurate results.

Knowledge of reference intervals for blood analytes, including serum protein fractions, is of great importance for the identification of infectious and inflammatory diseases and is often lacking in wild animal species. Serum samples were obtained from European minks enrolled in the breeding program (n = 55). Agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) were used to separate and identify protein fractions. Albumin, α1, α2, β, and γ-globulins fractions were identified in all mink sera by both electrophoresis methods. Reference intervals (90% CI) were determined following the 2008 guidelines of the Clinical Laboratory Standard Institute. The methods were compared using Passing-Bablok regression, Bland-Altman analysis, and Lin's concordance correlation. A significant bias was found between methods for α1, α2, and γ-globulin. Lin's concordance correlation was considered unacceptable for α1, α2, and β-globulins. Differences for gender between methods were found for albumin and α2-globuins, which were higher for males than females. γ-globulins were higher for adults than young minks using both methods; however, α1 and α2-globulins were lower. Both methods are adequate for identifying serum protein disorders, but the AGE and CZE methods are not equivalent. Therefore, reference intervals for each technique are required.

Candida albicans produces a complex glycosphingolipid called phospholipomannan (PLM), which is present on the cell-wall surface of yeast and shed upon contact with host cells. The glycan moiety of PLM is composed of β-mannosides with degrees of polymerization up to 19 in C. albicans serotype A. PLM from serotype B strains displays a twofold decrease in the length of the glycan chains. In this study we compared the proinflammatory activities of PLMs purified from C. albicans serotype A and serotype B strains and from a bmt6Δ mutant of C. albicans, whose PLM is composed of short truncated oligomannosidic chain. We found that PLMs activate caspase-1 in murine macrophage cell line J774 independent of the glycan chain length although IL-1β secretion is more intense with long glycan chain. None of the tested PLMs stimulate ROS production, indicating that caspase-1 activation may occur through a ROS-independent pathway. On the other hand, only long-chain oligomannosides present on PLM from serotype A strain (PLM-A) are able to induce TNF-α production in macrophages, a property that is not affect by blocking endocytosis through latrunculin A treatment. Finally, we demonstrate that soluble and not cell surface-bound galectin-3, is able to potentiate PLM-A-induced TNF-α production in macrophages. PLMs from C. albicans serotype B and from bmt6∆ mutant are not able to induce TNF-α production and galectin-3 pretreatment does not interfere with this result. In conclusion, we show here that PLMs are able to evoke a proinflammatory state in macrophage, which is in part dependent on their glycosylation status. Long-glycan chains favor interaction with soluble galectin-3 and help amplify inflammatory response.

Circulating tumor cells, a component of the “liquid biopsy”, hold great potential to transform the current landscape of cancer therapy. A key challenge to unlocking the clinical utility of CTCs lies in the ability to detect and isolate these rare cells using methods amenable to downstream characterization and other applications. In this review, we will provide an overview of current technologies used to detect and capture CTCs with brief insights into the workings of individual technologies. We focus on the strategies employed by different platforms and discuss the advantages of each. As our understanding of CTC biology matures, CTC technologies will need to evolve, and we discuss some of the present challenges facing the field in light of recent data encompassing epithelial-to-mesenchymal transition, tumor-initiating cells, and CTC clusters. •We present a comprehensive overview of CTC detection and capture technologies.•We provide a conceptual description of strategies used in different technologies.•We highlight the key features of individual technologies.•We discuss CTC technology performance in the context of clinical studies.

Glycosaminoglycans (GAGs) due to their hydrophilic character and high anionic charge densities play important roles in various (patho)physiological processes. The identification and quantification of GAGs in biological samples and tissues could be useful prognostic and diagnostic tools in pathological conditions. Despite the noteworthy progress in the development of sensitive and accurate methodologies for the determination of GAGs, there is a significant lack in methodologies regarding sample preparation and reliable fast analysis methods enabling the simultaneous analysis of several biological samples. In this report, developed protocols for the isolation of GAGs in biological samples were applied to analyze various sulfated chondroitin sulfate- and hyaluronan-derived disaccharides using fluorophore-assisted carbohydrate electrophoresis (FACE). Applications to biologic samples of clinical importance include blood serum, lens capsule tissue and urine. The sample preparation protocol followed by FACE analysis allows quantification with an optimal linearity over the concentration range 1.0–220.0 µg/mL, affording a limit of quantitation of 50 ng of disaccharides. Validation of FACE results was performed by capillary electrophoresis and high performance liquid chromatography techniques.

Background: The mucosa‐associated bacteria (MAB) are suspected of being involved in the pathogenesis of Crohn's disease. We analyzed and compared the MAB in noninflamed and inflamed ileal mucosa of Crohn's disease patients (n = 22). Methods: Tissue samples from the inflamed ileal mucosa and from the adjacent noninflamed ileal mucosa were taken from surgical resection specimens. The MAB were investigated using fluorescence in situ hybridization with 7 group‐specific probes and temporal temperature gradient gel electrophoresis (TTGE). Results: Samples from both noninflamed and inflamed mucosa were obtained from 15 patients. The distribution of the bacterial populations was not different between noninflamed and inflamed mucosa. The Bacteroidetes phylum was dominant and accounted for 29% of MAB (0%–74%) in noninflamed tissues and 32% (0%–70%) in inflamed areas. The γ Proteobacteria represented 12% (0%–70%) of MAB both in noninflamed and inflamed areas. The Clostridium coccoides group (Firmicutes phylum) represented 15% of MAB in noninflamed tissues versus 7% in inflamed areas. For most of the patients the similarity index between TTGE paired profiles was very high. Conclusion: The dominant MAB do not differ between noninflamed and inflamed ileal mucosa in Crohn's disease. This argues against a localized dysbiosis to explain the patchy distribution of mucosal lesions. (Inflamm Bowel Dis 2007)