Recombinant Flag-tagged E1E2 glycoproteins from three hepatitis C virus genotypes are biologically functional and elicit cross-reactive neutralizing antibodies in mice

Hepatitis C virus (HCV) is a globally disseminated human pathogen for which no vaccine is currently available. HCV is highly diverse genetically and can be classified into 7 genotypes and multiple sub-types. Due to this antigenic variation, the induction of cross-reactive and at the same time neutralizing antibodies is a challenge in vaccine production. Here we report the analysis of immunogenicity of recombinant HCV envelope glycoproteins from genotypes 1a, 1b and 2a, with a Flag tag inserted in the hypervariable region 1 of E2. This modification did not affect protein expression or conformation or its capacity to bind the crucial virus entry factor, CD81. Importantly, in immunogenicity studies on mice, the purified E2-Flag mutants elicited high-titer, cross-reactive antibodies that were able to neutralize HCV infectious particles from two genotypes tested (1a and 2a). These findings indicate that E1E2-Flag envelope glycoproteins could be important immunogen candidates for vaccine aiming to induce broad HCV-neutralizing responses

Computational methods for prediction of in vitro effects of new chemical structures

Background With a constant increase in the number of new chemicals synthesized every year, it becomes important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. In recent years, in silico prediction methods received great attention in an attempt to reduce animal experiments for the evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine. Various computational approaches have been proposed for the prediction of compound toxicity ranging from quantitative structure activity relationship modeling to molecular similarity-based methods and machine learning. Within the “Toxicology in the 21st Century” screening initiative, a crowd-sourcing platform was established for the development and validation of computational models to predict the interference of chemical compounds with nuclear receptor and stress response pathways based on a training set containing more than 10,000 compounds tested in high-throughput screening assays. Results Here, we present the results of various molecular similarity-based and machine-learning based methods over an independent evaluation set containing 647 compounds as provided by the Tox21 Data Challenge 2014. It was observed that the Random Forest approach based on MACCS molecular fingerprints and a subset of 13 molecular descriptors selected based on statistical and literature analysis performed best in terms of the area under the receiver operating characteristic curve values. Further, we compared the individual and combined performance of different methods. In retrospect, we also discuss the reasons behind the superior performance of an ensemble approach, combining a similarity search method with the Random Forest algorithm, compared to individual methods while explaining the intrinsic limitations of the latter. Conclusions Our results suggest that, although prediction methods were optimized individually for each modelled target, an ensemble of similarity and machine-learning approaches provides promising performance indicating its broad applicability in toxicity prediction

Lipofuscin, its origin, properties, and contribution to retinal fluorescence as a potential biomarker of oxidative damage to the retina

Lipofuscin accumulates with age as intracellular fluorescent granules originating from incomplete lysosomal digestion of phagocytosed and autophagocytosed material. The purpose of this review is to provide an update on the current understanding of the role of oxidative stress and/or lysosomal dysfunction in lipofuscin accumulation and its consequences, particularly for retinal pigment epithelium (RPE). Next, the fluorescence of lipofuscin, spectral changes induced by oxidation, and its contribution to retinal fluorescence are discussed. This is followed by reviewing recent developments in fluorescence imaging of the retina and the current evidence on the prognostic value of retinal fluorescence for the progression of age-related macular degeneration (AMD), the major blinding disease affecting elderly people in developed countries. The evidence of lipofuscin oxidation in vivo and the evidence of increased oxidative damage in AMD retina ex vivo lead to the conclusion that imaging of spectral characteristics of lipofuscin fluorescence may serve as a useful biomarker of oxidative damage, which can be helpful in assessing the efficacy of potential antioxidant therapies in retinal degenerations associated with accumulation of lipofuscin and increased oxidative stress. Finally, amendments to currently used fluorescence imaging instruments are suggested to be more sensitive and specific for imaging spectral characteristics of lipofuscin fluorescence

DYNAMIC OVERLOADS IN SELECTED GYMNASTIC EXERCISES

The purpose of this study is to identify force and time characteristics of ground reaction forces produced in selected movement exercises of gymnastics that contain elements of take-off and landing. The study was conducted over four athletes, two of sports acrobatics (17 and 18 yo) and two of artistic gymnastics (10 and 11 yo). The vertical ground reaction forces recorded in time were used to calculate maximum force values and characteristic times, and permitted an evaluation of the degree of dynamic overloads. The results of the tests show high levels of dynamic overloads, which may have traumatogenic character

Free Asynchronous Professional Development By, From, and For Instructional Designers: How Informal Learning Opportunities Shape Our Professional Learning and Design Practices

Instructional designers (IDs) need to maintain an understanding of the current trends and issues within the field. Pursuing professional learning informally supports IDs’ effort to keep up with current trends and issues because it is not restricted by curriculum and time. Professional development (PD) offered by Professional Development for Instructional Designers (PD4IDs) learning group can address issues related to geographical and funding limitations. This application paper presents the coordination of PD based on the conceptual framework (e.g., Community of Practice and Social Network Knowledge Construction) and reflections of several PD4IDs members with various roles. The reflections indicate the benefits of participating in PD for shaping IDs’ professional learning and practices. Discussion and implications for IDs intending to pursue non-traditional PD are also presented

Photodegradation of lipofuscin in suspension and in ARPE-19 cells and the similarity of fluorescence of the photodegradation product with oxidized docosahexaenoate

Retinal lipofuscin accumulates with age in the retinal pigment epithelium (RPE), where its fluorescence properties are used to assess retinal health. It was observed that there is a decrease in lipofuscin fluorescence above the age of 75 years and in the early stages of age-related macular degeneration (AMD). The purpose of this study was to investigate the response of lipofuscin isolated from human RPE and lipofuscin-laden cells to visible light, and to determine whether an abundant component of lipofuscin, docosahexaenoate (DHA), can contribute to lipofuscin fluorescence upon oxidation. Exposure of lipofuscin to visible light leads to a decrease in its long-wavelength fluorescence at about 610 nm, with a concomitant increase in the short-wavelength fluorescence. The emission spectrum of photodegraded lipofuscin exhibits similarity with that of oxidized DHA. Exposure of lipofuscin-laden cells to light leads to a loss of lipofuscin granules from cells, while retaining cell viability. The spectral changes in fluorescence in lipofuscin-laden cells resemble those seen during photodegradation of isolated lipofuscin. Our results demonstrate that fluorescence emission spectra, together with quantitation of the intensity of long-wavelength fluorescence, can serve as a marker useful for lipofuscin quantification and for monitoring its oxidation, and hence useful for screening the retina for increased oxidative damage and early AMD-related changes

Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Chatelier's Principle

Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect

Mixed-meal tolerance test to assess residual beta-cell secretion: Beyond the area-under-curve of plasma C-peptide concentration.

AIMS: Residual beta-cell secretion in type 1 diabetes is commonly assessed by area-under-curve of plasma C-peptide concentration (AUCCpep ) following mixed-meal tolerance test (MMTT). We aimed to investigate alternative measures of beta-cell responsiveness. METHODS: We analyzed data from 32 youth (age 7 to 17 years) undergoing MMTT within 6 months of type 1 diabetes diagnosis. We related AUCCpep with (a) validated mechanistic index of postprandial beta-cell responsiveness MI accounting for glucose level during MMTT, and (b) pragmatic marker calculated as baseline plasma C-peptide concentration corrected for baseline plasma glucose concentration. RESULTS: Postprandial responsiveness MI was correlated with age and BMI SDS (Rs = 0.66 and 0.44, P < 0.01 and P < 0.05) and was more correlated with glycated hemoglobin than AUCCpep (Rs = 0.79, P = 0.04). The pragmatic marker was highly correlated with AUCCpep (Rs = 0.94, P < 0.01). CONCLUSIONS: Postprandial responsiveness MI may be more relevant to glucose control than AUCCpep . Baseline C-peptide corrected for baseline glucose appears to be a suitable surrogate of AUCCpep if MMTT is not performed