The cell cycle program of polypeptide labeling in Chlamydomonas reinhardtii

The cell cycle program of polypeptide labeling in syndhronous cultures of wild-type Chlamydomonas reinhardtii was analyzed by pulse-labeling cells with 35SO4 = or [3H]arginine at different cell cycle stages. Nearly 100 labeled membrane and soluble polypeptides were resolved and studied using one-dimensional sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis. The labeling experiments produced the following results. (a) Total 35SO4 = and [3H]arginine incorporation rates varied independently throughout the cell cycle. 35SO4 = incorporation was highest in the mid-light phase, while [3H]arginine incorporation peaked in the dark phase just before cell division. (b) The relative labeling rate for 20 of 100 polypeptides showed significant fluctuations (3-12 fold) during the cell cycle. The remaining polypeptides were labeled at a rate commensurate with total 35SO4 = or [3H]arginine incorporation. The polypeptides that showed significant fluctuations in relative labeling rates served as markers to identify cell cycle stages. (c) The effects of illumination conditions on the apparent cell cycle stage-specific labeling of polypeptides were tested. Shifting light-grown asynchronous cells to the dark had an immediate and pronounced effect on the pattern of polypeptide labeling, but shifting dark-phase syndhronous cells to the light had little effect. The apparent cell cycle variations in the labeling of ribulose 1,5-biphosphate (RUBP)-carboxylase were strongly influenced by illumination effects. (d) Pulse-chase experiments with light-grown asynchronous cells revealed little turnover or inter- conversion of labeled polypeptides within one cell generation, meaning that major polypeptides, whether labeled in a stage-specific manner or not, do not appear transiently in the cell cycle of actively dividing, light-grown cells. The cell cycle program of labeling was used to analyze effects of a temperature-sensitive cycle blocked (cb) mutant. A synchronous culture of ts10001 was shifted to restrictive temperature before its block point to prevent it from dividing. The mutant continued its cell cycle program of polypeptide labeling for over a cell generation, despite its inability to divide

Three-Dimensional Motion Analysis for Occupational Therapy Upper Extremity Assessment and Rehabilitation: A Scoping Review

Background: Three-dimensional (3D) human motion analysis provides objective, quantitative, and reliable kinematic data that are valuable in rehabilitation. Clinicians, including occupational therapists and other specialists, can apply this technology to quantify patients’ upper extremity (UE) motion during functional tasks. A better comprehension of altered body mechanics serves to guide clinical reasoning, develop evidence-based interventions, and monitor patients’ progress through follow-up. However, the scientific literature has yet to emphasize the practicality of using 3D motion analysis as a clinical measurement tool. Method: This scoping review appraised 20 articles that used 3D motion analysis to quantify UE movements for individuals with and without mechanical pathologies. The articles were evaluated based on their quality and clinically relevant applications of UE kinematics. Results: This scoping review revealed that 3D motion analysis has already been implemented in rehabilitation but the variability across protocols and facilities can complicate the comparison of results. Conclusion: To further expand clinical use of 3D motion analysis, an introduction of more accessible, inexpensive, and user-friendly kinematic systems is critical. Future research should also aim to establish a standardized protocol of 3D motion analysis in UE assessments to produce clinically relevant results and maximize patients’ independence when engaging in daily activities

Subdiffusive axial transport of granular materials in a long drum mixer

Granular mixtures rapidly segregate radially by size when tumbled in a partially filled horizontal drum. The smaller component moves toward the axis of rotation and forms a buried core, which then splits into axial bands. Models have generally assumed that the axial segregation is opposed by diffusion. Using narrow pulses of the smaller component as initial conditions, we have characterized axial transport in the core. We find that the axial advance of the segregated core is well described by a self-similar concentration profile whose width scales as $t^\alpha$, with $\alpha \sim 0.3 < 1/2$. Thus, the process is subdiffusive rather than diffusive as previously assumed. We find that $\alpha$ is nearly independent of the grain type and drum rotation rate within the smoothly streaming regime. We compare our results to two one-dimensional PDE models which contain self-similarity and subdiffusion; a linear fractional diffusion model and the nonlinear porous medium equation.Comment: 4 pages, 4 figures, 1 table. Submitted to Phys Rev Lett. For more info, see http://www.physics.utoronto.ca/nonlinear

Bi-Directional Exchange of Membrane Components Occurs during Co-Culture of Mesenchymal Stem Cells and Nucleus Pulposus Cells

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells

Determination of copper in tap water using solid-phase spectrophotometry

A new application of ion exchange films is presented. The films are used in a simple analytical method of directly determining low concentrations of Cu(2+) in aqueous solutions, in particular, drinking water. The basis for this new test method is the color and absorption intensity of the ion when adsorbed onto the film. The film takes on the characteristic color of the adsorbed cation, which is concentrated on the film by many orders of magnitude. The linear relationship between absorbance (corrected for variations in film thickness) and solution concentration makes the determinations possible. These determinations agree well with flame atomic absorption determinations

CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes

© 2020 The Authors G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels

Traveling Granular Segregation Patterns in a Long Drum Mixer

Mixtures of granular media often exhibit size segregation along the axis of a partially-filled, horizontal, rotating cylinder. Previous experiments have observed axial bands of segregation that grow from concentration fluctuations and merge in a manner analogous to spinodal decomposition. We have observed that a new dynamical state precedes this effect in certain mixtures: bi-directional traveling waves. By preparing initial conditions, we found that the wave speed decreased with wavelength. Such waves appear to be inconsistent with simple PDE models which are first order in time.Comment: 11 page

Using remote substituents to control solution structure and anion binding in lanthanide complexes.

A study of the anion-binding properties of three structurally related lanthanide complexes, which all contain chemically identical anion-binding motifs, has revealed dramatic differences in their anion affinity. These arise as a consequence of changes in the substitution pattern on the periphery of the molecule, at a substantial distance from the binding pocket. Herein, we explore these remote substituent effects and explain the observed behaviour through discussion of the way in which remote substituents can influence and control the global structure of a molecule through their demands upon conformational space. Peripheral modifications to a binuclear lanthanide motif derived from α,α′-bis(DO3 Ayl)-m-xylene are shown to result in dramatic changes to the binding constant for isophthalate. In this system, the parent compound displays considerable conformational flexibility, yet can be assumed to bind to isophthalate through a well-defined conformer. Addition of steric bulk remote from the binding site restricts conformational mobility, giving rise to an increase in binding constant on entropic grounds as long as the ideal binding conformation is not excluded from the available range of conformers

Bone density of first and second segments of normal and dysmorphic sacra

BACKGROUND: Iliosacral screw fixation is safe and effective but can be complicated by loss of fixation, particularly in patients with osteopenic bone. Sacral morphology dictates where iliosacral screws may be placed when stabilizing pelvic ring injuries. In dysmorphic sacra, the safe osseous corridor of the upper sacral segment (S1) is smaller and lacks a transsacral corridor, increasing the need for fixation in the second sacral segment (S2). Previous evidence suggests that S2 is less dense than S1. The aim of this cross-sectional study is to further evaluate bone mineral density (BMD) of the S1 and S2 iliosacral osseous pathways through morphology stratification into normal and dysmorphic sacra. MATERIALS AND METHODS: Pelvic computed tomography scans of 50 consecutive trauma patients, aged 18 to 50 years, from a level 1 trauma center were analyzed prospectively. Five radiographic features (upper sacral segment not recessed in the pelvis, mammillary bodies, acute alar slope, residual S1 disk, and misshapen sacral foramen) were used to identify dysmorphic characteristics, and sacra with four or five features were classified as dysmorphic. Hounsfield unit values were used to estimate the regional BMD of S1 and S2. Student\u27s t-test was utilized to compare the mean values at each segment, with statistical significance being set at p \u3c 0.05. No change in clinical management occurred as a result of inclusion in this study. RESULTS: A statistical difference in BMD was appreciated between S1 and S2 in both normal and dysmorphic sacra (p \u3c 0.0001), with 28.4% lower density in S2 than S1. Further, S1 in dysmorphic sacra tended to be 4% less dense than S1 in normal sacra (p = 0.047). No difference in density was appreciated at S2 based on morphology. CONCLUSIONS: Our results would indicate that, based on BMD alone, fixation should be maximized in S1 prior to fixation in S2. In cases where S2 fixation is required, we recommend that transsacral fixation should be strongly considered if possible to bypass the S2 body and achieve fixation in the cortical bone of the ilium and sacrum. LEVEL OF EVIDENCE: Level III

Transcriptional profiling of cattle infected with Trypanosoma congolense highlights gene expression signatures underlying trypanotolerance and trypanosusceptibility

<p>Abstract</p> <p>Background</p> <p>African animal trypanosomiasis (AAT) caused by tsetse fly-transmitted protozoa of the genus <it>Trypanosoma </it>is a major constraint on livestock and agricultural production in Africa and is among the top ten global cattle diseases impacting on the poor. Here we show that a functional genomics approach can be used to identify temporal changes in host peripheral blood mononuclear cell (PBMC) gene expression due to disease progression. We also show that major gene expression differences exist between cattle from trypanotolerant and trypanosusceptible breeds. Using bovine long oligonucleotide microarrays and real time quantitative reverse transcription PCR (qRT-PCR) validation we analysed PBMC gene expression in naïve trypanotolerant and trypanosusceptible cattle experimentally challenged with <it>Trypanosoma congolense </it>across a 34-day infection time course.</p> <p>Results</p> <p>Trypanotolerant N'Dama cattle displayed a rapid and distinct transcriptional response to infection, with a ten-fold higher number of genes differentially expressed at day 14 post-infection compared to trypanosusceptible Boran cattle. These analyses identified coordinated temporal gene expression changes for both breeds in response to trypanosome infection. In addition, a panel of genes were identified that showed pronounced differences in gene expression between the two breeds, which may underlie the phenomena of trypanotolerance and trypanosusceptibility. Gene ontology (GO) analysis demonstrate that the products of these genes may contribute to increased mitochondrial mRNA translational efficiency, a more pronounced B cell response, an elevated activation status and a heightened response to stress in trypanotolerant cattle.</p> <p>Conclusion</p> <p>This study has revealed an extensive and diverse range of cellular processes that are altered temporally in response to trypanosome infection in African cattle. Results indicate that the trypanotolerant N'Dama cattle respond more rapidly and with a greater magnitude to infection compared to the trypanosusceptible Boran cattle. Specifically, a subset of the genes analyzed by real time qRT-PCR, which display significant breed differences, could collectively contribute to the trypanotolerance trait in N'Dama.</p