Human α-fetoprotein as a Zn2+-binding protein. Tight cation binding is not accompanied by global changes in protein structure and stability

AbstractThe binding of zinc to human α-fetoprotein (AFP) isolated from human umbilical cord serum was studied by fluorimetric Zn2+-titration. We found that the total number of strong binding sites for zinc on this protein was 5: AFP has one very strong (dissociation constant, Kd<10−8 M) and at least four lower affinity zinc binding sites (Kd<10−5 M). Fourier transform infrared (FTIR) analysis revealed that aspartate and histidine residues could be involved in the strong coordination of zinc. Intriguingly, binding of zinc to the protein does not induce structural changes that can be detected by circular dichroism, FTIR, intrinsic fluorescence or (1,1′)-bi-(4-anilino)naphthalene-5,5′-disulfonic acid (bis-ANS) binding. Finally, scanning microcalorimetry measurements showed that stability of the protein is also unaffected by zinc binding in spite of the strength of the coordination. Such strong interactions without major structural consequences are highly unusual, and AFP may therefore be the first characterized representative of a new class of ligand-binding proteins

Enhancing the specific T cell immune response against micro- and nanoparticle immobilized antigen

The current study was a part of the project on generating viral particle traps occurring due to covalent immobilization on the interface of recombinant virus-specific polymer-based nano- and microparticles. It is assumed that protein-particle conjugates could be able to bind virions followed by engulfment by immune cells. The study was aimed to examine the effect of polylactic acid (PLA) and PLA block-copolymer with polyethylene glycol (PLA-PEG)-based micro- and nanoparticles on the cellular immune response against polymeric particle-bound antigen. Materials and methods. A recombinant chimeric protein beta-2-microglobulin — green fluorescent protein (β2M-sfGFP) was obtained by affine chromatography. The recombinant protein was immobilized onto the polymer particles, which were further used for mice immunization. Female F1 hybrid mice (CBA x C57BL) in experimental and control groups consisted of 4–6-month-old 15 animals (weighted 20–25 g). Intracellular cytokine staining was used to evaluate the cellular immune response. Results and discussion. It was shown that the nanoparticles of PLA block-copolymer with polyethylene glycol (PLA-PEG) were able to bind 10 microgram protein per 1 mg polymer. The polylactic acid nanoparticles were able to bind 2,3 microgram protein per 1 mg polymer. In experiment, mice in group 1 were immunized with 100 nm PLA-PEG particle-β2M-sfGFP conjugate, in group 2 — with same particles together with soluble β2M-sfGFP. In group 3, mice were immunized with 1400 nm PLA particles-β2M-sfGFP conjugate, and in group 4 — with same particles together with soluble protein. The spleens isolated 2 weeks after the four-time intraperitoneal immunization. Comparison of immune response between groups was assessed by nonparametric Kruskal–Wallis criterion with Tukey correction. It was shown that the number of antigen-specific CD4+ T cells produced to model protein was significantly higher after immunization with particle-β2M-sfGFP conjugate, as compared to control groups, wherein immunization was performed with a mixture of protein and unmodified particles (p &lt; 0.001). It was found that the number of antigen-specific CD8+ T cells formed against β2m-sfGFP did not differ between all groups examined

Dynamics of Peer Grading: An Empirical Study

Peer grading is widely used in MOOCs and in standard university settings. The quality of grades obtained via peer grading is essential for the educational process. In this work, we study the factors that influence errors in peer grading. We analyze 288 assignments with 25,633 submissions and 113,169 reviews conducted with CrowdGrader, a web based peer grading tool. First, we found that large grading errors are generally more closely correlated with hard-to-grade submission, rather than with imprecise students. Second, we detected a weak correlation between review accuracy and student proficiency, as measured by the quality of the student's own work. Third, we found little correlation between review accuracy and the time it took to perform the review, or how late in the review period the review was performed. Finally, we found a clear evidence of tit-for-tat behavior when students give feedback on the reviews they received. We conclude with remarks on how these data can lead to improvements in peer-grading tools

Dynamics of Peer Grading: An Empirical Study

Peer grading is widely used in MOOCs and in standard university settings. The quality of grades obtained via peer grading is essential for the educational process. In this work, we study the factors that influence errors in peer grading. We analyze 288 assignments with 25,633 submissions and 113,169 reviews conducted with CrowdGrader, a web based peer grading tool. First, we found that large grading errors are generally more closely correlated with hard-to-grade submission, rather than with imprecise students. Second, we detected a weak correlation between review accuracy and student proficiency, as measured by the quality of the student's own work. Third, we found little correlation between review accuracy and the time it took to perform the review, or how late in the review period the review was performed. Finally, we found a clear evidence of tit-for-tat behavior when students give feedback on the reviews they received. We conclude with remarks on how these data can lead to improvements in peer-grading tools

Reliable Aggregation of Boolean Crowdsourced Tasks

We propose novel algorithms for the problem of crowd- sourcing binary labels. Such binary labeling tasks are very common in crowdsourcing platforms, for instance, to judge the appropriateness of web content or to flag vandalism. We propose two unsupervised algorithms: one simple to implement albeit derived heuristically, and one based on iterated bayesian parameter estimation of user reputation models. We provide mathematical insight into the benefits of the proposed algorithms over existing approaches, and we confirm these insights by showing that both algorithms offer improved performance on many occasions across both synthetic and real-world datasets obtained via Amazon Mechanical Turk

Reliable Aggregation of Boolean Crowdsourced Tasks

We propose novel algorithms for the problem of crowd- sourcing binary labels. Such binary labeling tasks are very common in crowdsourcing platforms, for instance, to judge the appropriateness of web content or to flag vandalism. We propose two unsupervised algorithms: one simple to implement albeit derived heuristically, and one based on iterated bayesian parameter estimation of user reputation models. We provide mathematical insight into the benefits of the proposed algorithms over existing approaches, and we confirm these insights by showing that both algorithms offer improved performance on many occasions across both synthetic and real-world datasets obtained via Amazon Mechanical Turk

PLASMA CYTOKINES LEVELS IN PATIENTS UNDERGOING LONG-TERM HAEMODIALYSIS

Аbstract.  Patients  with  end-stage  renal  disease  need  their  kidney  functions  to  be  replaced.  Chronic haemodialysis represents a most common method of such substitution treatment. This procedure results in successful survival of such patients for years. Chronic haemodialysis is accompanied by a complication which is known as β2-microglobulin amyloidosis. In this case, amyloid substance consisting of β2-microglobulin (β2-MG) accumulates in bones, ligaments and joints. Biological causes of β2-MG amyloidosis are still not established. To elucidate the role of inflammation in the pathogenesis of β2-MG amyloidosis, the levels of  IL-2,  IL-4,  IL-6,  IL-8,  IL-10,  GM-CSF,  IFNγ, TNFα were quantified in plasma of patients undergoing  long-term haemodialysis. Mean amounts of all the mentioned cytokines in haemodialysis patients proved to be significantly higher than in control group consisting of healthy subjects. When comparing a group receiving standard  dialysis  procedure  versus  a  subgroup  receiving  haemodiafiltration,  a  single  reliable  difference  was revealed for GM-CSF levels (p &lt; 0.04), without any differences shown for other cytokines. With increasing terms of chronic haemodialysis, the levels of IL-2, IL-4, IL-6, IL-8, GM-CSF, IFNγ, TNFα were increased, or, at least, they did not decrease. After three years of dialysis, IL-10 concentrations were statistically indistinguishable from normal levels. In patients undergoing haemodiafiltration, plasma levels of IL-2, IL-4, IL-8, GM-CSF, IFNγ, TNFα did not drop with increasing terms of dialysis. The levels of IL-6 and IL-10 decreased after three years of dialysis, to near-normal levels.In general, these results suggest that IL-10 and IL-6 may be regarded as candidates for further studies as potential markers of β2-microglobulin amyloidosis. (Med. Immunol., 2011, vol. 13, N 2-3, pp 211-218

Biophysical Characterization of Albumin Preparations from Blood Serum of Healthy Donors and Patients with Renal Diseases. Part I: Spectrofluorometric Analysis

Background: Serum albumin (SA) is a major proteinacous component of the blood plasma. In the present study we report on the determination and structural characterization of the modified SA in the plasma of glomerulonephritis (GN) patients. Fluorescence analysis of albumin-enriched fractions (AEF) isolated from serum of GN patients leads to the discovery of a proteinacous component with essential blue shift of intrinsic fluorescence. Material/Methods: AEF were isolated from serum blood of 35 GN patients, 30 GN patients with chronic renal failure (CRF) corrected by hemodialysis (HD) and 40 healthy donors. Their fluorescence characteristics have been compared using variations in the value of parameter A = I320/I365 that characterizes fluorescence spectrum position and shape. Results: The fluorescence spectra of AEF from GN and GN with CRF patients were blue- and redshifted, respectively, in comparison with spectra of donors’ serum. The value of parameter A was 1.27±0.05 for protein preparations from healthy donors, while this characteristic lies within range of 1.3–2.1 or 0.77–1.29 for GN and GN with CRF patients, respectively. Moreover, for GN patients, the magnitude of spectral parameter A was approaching control values during the treatment of disease. Conclusion: hSA enriched fractions isolated from GN patients or GN patients with CRF corrected by HD differ by their spectrofluorometric properties from those of healthy donors. This technique allows the detection of renal diseases and the efficiency of their cure