Determination of soluble wheat protein fractions using the Bradford assay

Background and objectives Determination of different grain protein fractions in wheat cultivars is an important task in analyzing bread baking quality. In many laboratories, the Bradford assay is used to determine protein concentrations in solutions. In any protein assay (including Bradford), the ideal protein to use as a standard is the purified protein being assayed. In the absence of such an absolute reference, protein another protein must be selected as a relative standard such as bovine serum albumin (BSA) which is widely used. The aim of this work was to find conversion factors for BSA to determine correct albumin–globulin, gliadin, and glutenin concentrations, because these purified wheat grain protein fractions are mostly not available to be used for calibration purposes. Findings In case of BSA calibration, gluten concentration was underestimated (50%–54%) compared to calibration with the respective purified wheat proteins (65%–70%) in extracts of wheat grain samples. This result is explained with the different amino acid composition of BSA and wheat protein fractions leading to a more intense signal with BSA in the Bradford assay. Calibration of the Bradford assay using BSA as well as purified wheat protein fractions allowed to calculate the conversion factors of 2.11 for BSA/albumin–globulin, 4.25 for BSA/gliadin, and 3.42 for BSA/glutenin. Application of these conversion factors proved to accurately adjust protein concentrations of wheat fractions originating from ten cultivars, determined with BSA calibration of the Bradford assay. Conclusions BSA calibration of the Bradford assay in combination with the conversion factors can be used to determine protein concentration of wheat grain fractions. Significance and novelty Findings of this study make a contribution toward the correction of a common method, to provide a basis for better comparability of research results from different laboratories

TKA patients with unsatisfying knee function show changes in neuromotor synergy pattern but not joint biomechanics

Nearly 20% of patients who have undergone total knee arthroplasty (TKA) report persistent poor knee function. This study explores the idea that, despite similar knee joint biomechanics, the neuro-motor synergies may be different between high-functional and low-functional TKA patients. We hypothesized that (1) high-functional TKA recruit a more complex neuro-motor synergy pattern compared to low-functional TKA and (2) high-functional TKA patients demonstrate more stride-to-stride variability (flexibility) in their synergies. Gait and electromyography (EMG) data were collected during level walking for three groups of participants: (i) high-functional TKA patients (n = 13); (ii) low-functional TKA patients (n = 13) and (iii) non-operative controls (n = 18). Synergies were extracted from EMG data using non-negative matrix factorization. Analysis of variance and Spearman correlation analyses were used to investigate between-group differences in gait and neuro-motor synergies. Results showed that synergy patterns were different among the three groups. Control subjects used 5–6 independent neural commands to execute a gait cycle. High functional TKA patients used 4–5 independent neural commands while low-functional TKA patients relied on only 2–3 independent neural commands to execute a gait cycle. Furthermore, stride-to-stride variability of muscles’ response to the neural commands was reduced up to 15% in low-functional TKAs compared to the other two groups

Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters

<p>Abstract</p> <p>Background</p> <p>The red flour beetle <it>Tribolium castaneum </it>has developed into an insect model system second only to <it>Drosophila</it>. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for <it>Tribolium </it>research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.</p> <p>Results</p> <p>Here we report the establishment of the GAL4/UAS binary expression system in <it>Tribolium castaneum</it>. Both GAL4Δ and GAL4VP16 driven by the endogenous heat shock inducible promoter of the <it>Tribolium hsp68 </it>gene are efficient in activating reporter gene expression under the control of the Upstream Activating Sequence (UAS). UAS driven ubiquitous tGFP fluorescence was observed in embryos within four hours after activation while <it>in-situ </it>hybridization against tGFP revealed expression already after two hours. The response is quick in relation to the duration of embryonic development in <it>Tribolium </it>- 72 hours with segmentation being completed after 24 hours - which makes the study of early embryonic processes possible using this system. By comparing the efficiency of constructs based on <it>Tribolium, Drosophila</it>, and artificial core promoters, respectively, we find that the use of endogenous core promoters is essential for high-level expression of transgenic constructs.</p> <p>Conclusions</p> <p>With the established GAL4/UAS binary expression system, ectopic misexpression approaches are now feasible in <it>Tribolium</it>. Our results support the contention that high-level transgene expression usually requires endogenous regulatory sequences, including endogenous core promoters in <it>Tribolium </it>and probably also other model systems.</p

Influence of head size on the development of metallic wear and on the characteristics of carbon layers in metal-on-metal hip joints

Background and purpose Particles originating from the articulating surfaces of hip endoprostheses often induce an inflammatory response, which can be related to implant failure. We therefore analyzed the metal content in capsular tissue from 44 McKee-Farrar metal-on-metal hip prostheses (with 3 different head sizes) and we also analyzed the morphological structure of layers located on articulating surfaces

Measuring multipartite entanglement via dynamic susceptibilities

Entanglement plays a central role in our understanding of quantum many body physics, and is fundamental in characterising quantum phases and quantum phase transitions. Developing protocols to detect and quantify entanglement of many-particle quantum states is thus a key challenge for present experiments. Here, we show that the quantum Fisher information, representing a witness for genuinely multipartite entanglement, becomes measurable for thermal ensembles via the dynamic susceptibility, i.e., with resources readily available in present cold atomic gas and condensed-matter experiments. This moreover establishes a fundamental connection between multipartite entanglement and many-body correlations contained in response functions, with profound implications close to quantum phase transitions. There, the quantum Fisher information becomes universal, allowing us to identify strongly entangled phase transitions with a divergent multipartiteness of entanglement. We illustrate our framework using paradigmatic quantum Ising models, and point out potential signatures in optical-lattice experiments.Comment: 5+5 pages, 3+2 figure

European multidisciplinary consensus statement on the use and monitoring of metal-on-metal bearings for total hip replacement and hip resurfacing.

Summary Introduction There is an ongoing debate about the optimal use of metal-on-metal (MoM) bearings in total hip replacement, since there are uncertainties about local and systemic adverse effects due to wear and corrosion of these bearings. Despite various national recommendations, efforts to achieve international harmonization of specific evidence-based recommendations for best practice are still lacking. Hypothesis An international consensus study group should be able to develop recommendations on the use and monitoring of MoM bearings, preferably at the European level, through a multidisciplinary approach, by integrating the perspectives of various stakeholders. Materials and methods Twenty-one experts representing three stakeholder groups and eight countries participated in this European consensus study, which consisted of a consensus meeting, subsequent structured discussion, and consensus voting. Results The current statement defines first of all benefits, local and systemic risks, as well as uncertain issues related to MoM bearings. Safety assessment after implantation of MoM comprises all patients. A closer follow-up is recommended for large head MoM (≥ 36 mm) and resurfacing. In these implants basic follow-up should consist of x-rays and metal ion measurement of cobalt in whole blood, performed with GF-AAS or ICP-MS. Clinical and/or radiographic abnormality as well as elevated ion levels needs additional imaging (ultrasound, CT-scan and/or MARS-MRI). Cobalt values less than 2 μg/L are probably devoid of clinical concern, the threshold value for clinical concern is expected to be within the range of 2–7 μg/L. Discussion This is the first multinational, interdisciplinary, and multiprofessional approach for developing a recommendation for the use and monitoring of MoM bearings in total hip replacement. The current recommendations are in partial agreement with previous statements regarding the extent of follow-up and imaging techniques. They however differ from previous communications regarding measurement of metal ions and especially the investigated medium, technique, and eventual threshold levels. Level of evidence Level V, expert opinion/agreement conference

Validation Study for Non-Invasive Prediction of IDH Mutation Status in Patients with Glioma Using In Vivo 1H-Magnetic Resonance Spectroscopy and Machine Learning

The isocitrate dehydrogenase (IDH) mutation status is an indispensable prerequisite for diagnosis of glioma (astrocytoma and oligodendroglioma) according to the WHO classification of brain tumors 2021 and is a potential therapeutic target. Usually, immunohistochemistry followed by sequencing of tumor tissue is performed for this purpose. In clinical routine, however, non-invasive determination of IDH mutation status is desirable in cases where tumor biopsy is not possible and for monitoring neuro-oncological therapies. In a previous publication, we presented reliable prediction of IDH mutation status employing proton magnetic resonance spectroscopy (1H-MRS) on a 3.0 Tesla (T) scanner and machine learning in a prospective cohort of 34 glioma patients. Here, we validated this approach in an independent cohort of 67 patients, for which 1H-MR spectra were acquired at 1.5 T between 2002 and 2007, using the same data analysis approach. Despite different technical conditions, a sensitivity of 82.6% (95% CI, 61.2-95.1%) and a specificity of 72.7% (95% CI, 57.2-85.0%) could be achieved. We concluded that our 1H-MRS based approach can be established in a routine clinical setting with affordable effort and time, independent of technical conditions employed. Therefore, the method provides a non-invasive tool for determining IDH status that is well-applicable in an everyday clinical setting

Patient and surgery related factors associated with fatigue type polyethylene wear on 49 PCA and DURACON retrievals at autopsy and revision

<p>Abstract</p> <p>Background</p> <p>Polyethylene wear is an important factor for longevity of total knee arthroplasty. Proven and suspicious factors causing wear can be grouped as material, patient and surgery related. There are more studies correlating design and/or biomaterial factors to in vivo wear than those to patient and surgery related factors. Many retrieval studies just include revision implants and therefore may not be representative. This study is aimed to correlate patient- and surgery- related factors to visual wear score by minimizing design influence and include both autopsy and revision implants. Comparison between the groups was expected to unmask patient and surgery-related factors responsible for wear.</p> <p>Methods</p> <p>The amount of joint side wear on polyethylene retrievals was measured using a modification of an established visual wear score. Fatigue type wear was defined as summation of the most severe wear modes of delamination, pitting and cracks. Analysis of patient and surgery related variables suspicious to cause wear included prospectively sampled patient activity which was measured by self reported walking capacity. Statistical analysis was done by univariate analysis of variance. Activity level and implantation time were merged to an index of use and correlated to the wear score.</p> <p>Results</p> <p>Wear score after comparable implantation time was significantly less in the autopsy group. Even so, fatigue type wear accounted for 84 and 93 % of total wear score on autopsy and revision implants respectively. A highly significant influence on wear score was found in time of implantation (p = 0.002), level of activity (p = 0.025) and inserts belonging to revision group (p = 0.006). No influence was found for the kind of patella replacement (p = 0.483). Body mass index and accuracy of component alignment had no significant influence on visual wear score. Fatigue-type wear in the medial compartment was closely correlated to the index of use in the autopsy (R²= 0.383) and the revision group (R²= 0.813).</p> <p>Conclusion</p> <p>The present study's finding of substantial fatigue type wear in both autopsy and revision retrievals supports the theory that polyethylene fatigue strength is generally exceeded in this type of prosthesis. Furthermore, this study correlated fatigue-type polyethylene wear to an index of use as calculated by activity over time. Future retrieval studies may use activity over time as an important patient related factor correlated to the visual wear score. When evaluating total knee arthroplasty routine follow up, the surgeon must think of substantial wear present even without major clinical signs.</p

Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 µm×50 µm×2.5 µm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy

Pathogenic T cell responses against aquaporin 4

Inflammatory lesions in the central nervous system of patients with neuromyelitis optica are characterized by infiltration of T cells and deposition of aquaporin-4-specific antibodies and complement on astrocytes at the glia limitans. Although the contribution of aquaporin-4-specific autoantibodies to the disease process has been recently elucidated, a potential role of aquaporin-4-specific T cells in lesion formation is unresolved. To address this issue, we raised aquaporin-4-specific T cell lines in Lewis rats and characterized their pathogenic potential in the presence and absence of aquaporin-4-specific autoantibodies of neuromyelitis optica patients. We show that aquaporin-4-specific T cells induce brain inflammation with particular targeting of the astrocytic glia limitans and permit the entry of pathogenic anti-aquaporin-4-specific antibodies to induce NMO-like lesions in spinal cord and brain. In addition, transfer of aquaporin-4-specific T cells provoked mild (subclinical) myositis and interstitial nephritis. We further show that the expression of the conformational epitope, recognized by NMO patient-derived aquaporin-4-specific antibodies is induced in kidney cells by the pro-inflammatory cytokine gamma-interferon. Our data provide further support for the view that NMO lesions may be induced by a complex interplay of T cell mediated and humoral immune responses against aquaporin-4