Thin films of Type 1 collagen for cell by cell analysis of morphology and tenascin-C promoter activity

BACKGROUND: The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of cells on different extracellular matrices. We have quantitatively examined the relationship between fibroblast morphology and activation of the promoter for the extracellular matrix protein tenascin-C using a tenascin-C promoter-based GFP reporter construct. RESULTS: We find that when considering the average response from the population of cells, cell area correlates with tenascin-C promoter activity as has been previously suggested; however cell-by-cell analysis suggests that cell area and promoter activity are not tightly correlated within individual cells. CONCLUSION: This study demonstrates how quantitative cell-by-cell analysis, facilitated by the use of thin films of extracellular matrix proteins, can provide insight into the relationship between phenotypic parameters

Piping plovers demonstrate regional differences in nesting habitat selection patterns along the U. S. Atlantic coast

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Zeigler, S. L., Gutierrez, B. T., Hecht, A., Plant, N. G., & Sturdivant, E. J. Piping plovers demonstrate regional differences in nesting habitat selection patterns along the U. S. Atlantic coast. Ecosphere, 12(3), (2021): e03418, https://doi.org/10.1002/ecs2.3418.Habitat studies that encompass a large portion of a species’ geographic distribution can explain characteristics that are either consistent or variable, further informing inference from more localized studies and improving management successes throughout the range. We identified landscape characteristics at Piping Plover nests at 21 sites distributed from Massachusetts to North Carolina and compared habitat selection patterns among the three designated U.S. recovery units (New England, New York–New Jersey, and Southern). Geomorphic setting, substrate type, and vegetation type and density were determined in situ at 928 Piping Plover nests (hereafter, used resource units) and 641 random points (available resource units). Elevation, beach width, Euclidean distance to ocean shoreline, and least-cost path distance to low-energy shorelines with moist substrates (commonly used as foraging habitat) were associated with used and available resource units using remotely sensed spatial data. We evaluated multivariate differences in habitat selection patterns by comparing recovery unit-specific Bayesian networks. We then further explored individual variables that drove disparities among Bayesian networks using resource selection ratios for categorical variables and Welch’s unequal variances t-tests for continuous variables. We found that relationships among variables and their connections to habitat selection were similar among recovery units, as seen in commonalities in Bayesian network structures. Furthermore, nesting Piping Plovers consistently selected mixed sand and shell, gravel, or cobble substrates as well as areas with sparse or no vegetation, irrespective of recovery unit. However, we observed significant differences among recovery units in the elevations, distances to ocean, and distances to low-energy shorelines of used resource units. Birds also exhibited increased selectivity for overwash habitats and for areas with access to low-energy shorelines along a latitudinal gradient from north to south. These results have important implications for conservation and management, including assessment of shoreline stabilization and habitat restoration planning as well as forecasting effects of climate change.Funding for this work was provided by the North Atlantic Landscape Conservation Cooperative and U.S. Fish and Wildlife Service through a U.S. Geological Survey Mendenhall Fellowship to Zeigler. All other funding was through the U.S. Geological Survey (Zeigler, Gutierrez, Plant, and Sturdivant) and the U.S. Fish and Wildlife Service (Hecht). Zeigler, Plant, and Hecht conceived and designed the study and secured funding

Chromatographic Separation Apparatus

An apparatus for the monitoring of a column chromatography separation process includes a segmented column with a seal positioned at the joint defined by the segments of the column. A connector is provided for connecting the segments of the column together. The apparatus further includes a sensor for monitoring an analyte in an eluant within a separation zone of the column. The sensor includes a mesh grid made of optical fibers or metal wires which is placed so as to extend through the separation zone of the column. The metal wires or optical fibers extend through the seal of the joint in the segmented column and connect to signal processing and data analysis equipment for purposes of monitoring the movements and concentration of an analyte in an eluant at various locations within the column. Certain segments of the optical fibers or metal wires which make up the mesh grid are coated so as to be desensitized and other segments are uncoated for sensing the analyte. This provides an effective apparatus to monitor in detail the cross-section of a column chromatography process in-situ

Long-term stability of anti-cyclic citrullinated peptide antibody status in patients with early inflammatory polyarthritis.

INTRODUCTION: The utility of reassessing anti-cyclic citrullinated peptide (anti-CCP) antibody status later in disease in patients presenting with early undifferentiated inflammatory polyarthritis, particularly in those who test negative for both anti-CCP and rheumatoid factor (RF) at baseline, remains unclear. We aimed therefore to determine the stability of CCP antibody status over time and the prognostic utility of repeated testing in subjects with early inflammatory polyarthritis (IP). METHODS: Anti-CCP and RF were measured at baseline and 5 years in 640 IP patients from the Norfolk Arthritis Register, a primary care-based inception cohort. The relation between change in anti-CCP status/titer and the presence of radiologic erosions, the extent of the Larsen score, and Health Assessment Questionnaire (HAQ) score by 5 years was investigated. RESULTS: With a cut-off of 5 U/ml, 28% subjects tested positive for anti-CCP antibodies, 29% for RF, and 21% for both at baseline. Nine (2%) anti-CCP-negative patients seroconverted to positive, and nine (4.6%) anti-CCP-positive individuals became negative between baseline and 5 years. In contrast, RF status changed in 17% of subjects. However, change in RF status was strongly linked to baseline anti-CCP status and was not independently associated with outcome. Ever positivity for anti-CCP antibodies by 5 years did not improve prediction of radiographic damage compared with baseline status alone (accuracy, 75% versus 74%). A higher baseline anti-CCP titer (but not change in anti-CCP titer) predicted worse radiologic damage at 5 years (P < 0.0001), even at levels below the cut-off for anti-CCP positivity. Thus, a titer of 2 to 5 U/ml was strongly associated with erosions by 5 years (odds ratio, 3.6 (1.5 to 8.3); P = 0.003). CONCLUSIONS: Repeated testing of anti-CCP antibodies or RF in patients with IP does not improve prognostic value and should not be recommended in routine clinical practice.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

High frequency of antidrug antibodies and association of random drug levels with efficacy in certolizumab pegol-treated patients with rheumatoid arthritis: results from the BRAGGSS cohort

Objectives: To evaluate (i) the association between random certolizumab drug levels, antidrug antibodies (ADAbs) and treatment response in patients with rheumatoid arthritis (RA); (ii) longitudinal factors associated with ADAbs and certolizumab drug levels. Methods: This prospective cohort included 115 patients with RA treated with certolizumab. Serum samples were collected at 3, 6 and 12 months following treatment initiation. Drug levels and ADAbs were measured using ELISA and radioimmunoassay, respectively, at 3, 6 and 12 months. Disease Activity Score in 28 joints (DAS28) were measured at each visit and 12 months European League Against Rheumatism (EULAR) response was calculated. Patient self-reported adherence was collected longitudinally. Ordinal logistic regression and generalised estimating equation were used to test the association: (i) between drug levels, from serum sampled and treatment response; (ii) between ADAbs and drug levels; (iii) patient-centred factors and drug levels. Results: ADAbs were detected in 37% (42/112 patients by 12 months). The presence of ADAbs were significantly associated with lower drug levels over 12 months (β=−0.037, 95% CI −0.055 to 0.018, p<0.0001) but not independently with 12 months EULAR response (β=0.0013 (95% CI −0.0032 to 0.00061), p=0.18). Drug level was associated with 12 months EULAR response (β=0.032 (95% CI 0.0011 to 0.063), p=0.042). In the multivariate model, ADAb level and adherence were significantly associated with drug concentrations. Conclusions: This is the first study to demonstrate that higher certolizumab drug levels are associated with better 12 months EULAR response. ADAbs in certolizumab-treated patients with RA were detected at higher levels than previous studies and help determine the aetiology of a low drug level

Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy

<p>Abstract</p> <p>Background</p> <p>In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells.</p> <p>Results</p> <p>Paraformaldehyde fixation and Triton X-100 permeabilization preserved cell morphology and myosin light chain phosphorylation better than the alternative fixation/permeabilization methods tested. We utilized automated microscopy methods to acquire three color images, determine cell spread area, and quantify the intensity of staining within each cell with anti-phospho-MLC antibody. Our results indicate that A10 rat aortic smooth muscle cells exhibit a re producible non-Gaussian distribution of MLC phosphorylation across a population of unsynchronized genetically identical cells. Adding an inhibitor of Rho kinase, Y27632, or plating cells on a low density of fibronectin, reduced phospho-myosin light chain signal as expected. On the other hand, adding calyculin A, an activator of contractility, increased myosin light chain phosphorylation. The IC₅₀for myosin light chain phosphorylation using Y27632 was determined to be 2.1 ± 0.6 micrometers. We observed a positive linear relationship between cell area and myosin light chain diphosphorylation, which is consistent with what has been reported in the literature using other methods.</p> <p>Conclusion</p> <p>Our results show that using proper specimen fixation techniques and background subtraction methods, imaging cytometry can be used to reliably measure relative myosin light chain phosphorylation in individual adherent cells. Importantly, the ability to make this measurement in adherent cells allows for simultaneous measurement of and correlation with other parameters of cellular topography such as morphology and cell-cell proximity. This assay has potential application in screening for drug development.</p

Correction to: Transcriptome-wide study of TNF-inhibitor therapy in rheumatoid arthritis reveals early signature of successful treatment

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