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A correction to the article "Kinetochore components are required for central spindle assembly" in the April 13, 2015 issue is presented.

The objective of this study was to investigate whether 222Rn in groundwater can be used as a tracer for light non-aqueous phase liquid ( LNAPL) quantification at a field site treated by dual-phase LNAPL removal. After the break of a pipeline, 5 ha of soil in the nature reserve Coussouls de Crau in southern France was contaminated by 5100 m3 of crude oil. Part of this oil seeped into the underlying gravel aquifer and formed a floating oil body of about 3.9 ha. The remediation consists of plume management by hydraulic groundwater barriers and LNAPL extraction in the source zone. 222Rn measurements were performed in 21 wells in and outside the source zone during 15 months. In uncontaminated groundwater, the radon activity was relatively constant and remained always >11 Bq/L. The variability of radon activity measurements in wells affected by the pump-and-skim system was consistent with the measurements in wells that were not impacted by the system. The mean activities in wells in the source zone were, in general, significantly lower than in wells upgradient of the source zone, owing to partitioning of 222Rn into the oil phase. The lowest activities were found in zones with high non-aqueous phase liquid ( NAPL) recovery. LNAPL saturations around each recovery well were furthermore calculated during a period of high groundwater level, using a laboratory-determined crude oil-water partitioning coefficient of 38.5 ± 2.9. This yielded an estimated volume of residual crude oil of 309 ± 93 m3 below the capillary fringe. We find that 222Rn is a useful and cheap groundwater tracer for finding zones of good LNAPL recovery in an aquifer treated by dual-phase LNAPL removal, but that quantification of NAPL saturation using Rn is highly uncertain. [ABSTRACT FROM AUTHOR]

Cytokinesis, the physical division of one cell into two, is typically assumed to use the same molecular process across animal cells. However, regulation of cell division can vary significantly among different cell types, even within the same multicellular organism. Using six fast-acting temperature-sensitive (ts) cytokinesis-defective mutants, we found that each had unique cell type-specific profiles in the early C. elegans embryo. Certain cell types were more sensitive than others to actomyosin and spindle signaling disruptions, disrupting two members of the same complex could result in different phenotypes, and protection against actomyosin inhibition did not always protect against spindle signaling inhibition.

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab prophylaxis led us to employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We therefore analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab than in the presence of rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab than as for rFVIII/rFVIIIFc. Scanning electron microscopy and immunofluorescent spinning-disk confocal microscopy of in vivo-generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFcand emicizumab-induced clots. Contrary to the regular fibrin mesh obtained with rFVIII/rFVIIIFc, fibrin fibers appeared to be fused into large patches upon emicizumab treatment. Moreover, fewer red blood cells were detected in regions in which these fibrin patches were present. The presence of highly dense fibrin structures associated with a diffuse fiber structure in emicizumab-induced clots was also observed when using super-resolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Despite the crucial role of the extracellular matrix (ECM) in the organotypic organization and function of skeletal muscles, most 3D models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy, and porosity. Here, a novel 3D in vitro model of muscle ECM was developed reproducing these four crucial characteristics of the native ECM. An anisotropic hydrogel mimicking the muscle fascia was obtained thanks to unidirectional 3D printing of dense collagen with aligned collagen fibrils. The space between the different layers was tuned to generate an intrinsic network of pores (100 μm) suitable for nutrient and oxygen diffusion. By modulating the gelling conditions, the mechanical properties of the construct reached those measured in the physiological muscle ECM. This artificial matrix was thus evaluated for myoblast differentiation. The addition of large channels (600 μm) by molding permitted to create a second range of porosity suitable for cell colonization without altering the physical properties of the hydrogel. Skeletal myoblasts embedded in Matrigel®, seeded within the channels, organized in 3D, and differentiated into multinucleated myotubes. These results show that porous and anisotropic dense collagen hydrogels are promising biomaterials to model skeletal muscle ECM., Competing Interests: Declaration of competing interest The authors declare no conflict of interests relevant to this work., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Purpose: Heating of gradient coils and passive shim components is a common cause of instability in the B 0 field, especially when gradient intensive sequences are used. The aim of the study was to set a benchmark for typical drift encountered during MR spectroscopy (MRS) to assess the need for real-time field-frequency locking on MRI scanners by comparing field drift data from a large number of sites., Method: A standardized protocol was developed for 80 participating sites using 99 3T MR scanners from 3 major vendors. Phantom water signals were acquired before and after an EPI sequence. The protocol consisted of: minimal preparatory imaging; a short pre-fMRI PRESS; a ten-minute fMRI acquisition; and a long post-fMRI PRESS acquisition. Both pre- and post-fMRI PRESS were non-water suppressed. Real-time frequency stabilization/adjustment was switched off when appropriate. Sixty scanners repeated the protocol for a second dataset. In addition, a three-hour post-fMRI MRS acquisition was performed at one site to observe change of gradient temperature and drift rate. Spectral analysis was performed using MATLAB. Frequency drift in pre-fMRI PRESS data were compared with the first 5:20 minutes and the full 30:00 minutes of data after fMRI. Median (interquartile range) drifts were measured and showed in violin plot. Paired t-tests were performed to compare frequency drift pre- and post-fMRI. A simulated in vivo spectrum was generated using FID-A to visualize the effect of the observed frequency drifts. The simulated spectrum was convolved with the frequency trace for the most extreme cases. Impacts of frequency drifts on NAA and GABA were also simulated as a function of linear drift. Data from the repeated protocol were compared with the corresponding first dataset using Pearson's and intraclass correlation coefficients (ICC)., Results: Of the data collected from 99 scanners, 4 were excluded due to various reasons. Thus, data from 95 scanners were ultimately analyzed. For the first 5:20 min (64 transients), median (interquartile range) drift was 0.44 (1.29) Hz before fMRI and 0.83 (1.29) Hz after. This increased to 3.15 (4.02) Hz for the full 30 min (360 transients) run. Average drift rates were 0.29 Hz/min before fMRI and 0.43 Hz/min after. Paired t-tests indicated that drift increased after fMRI, as expected (p < 0.05). Simulated spectra convolved with the frequency drift showed that the intensity of the NAA singlet was reduced by up to 26%, 44 % and 18% for GE, Philips and Siemens scanners after fMRI, respectively. ICCs indicated good agreement between datasets acquired on separate days. The single site long acquisition showed drift rate was reduced to 0.03 Hz/min approximately three hours after fMRI., Discussion: This study analyzed frequency drift data from 95 3T MRI scanners. Median levels of drift were relatively low (5-min average under 1 Hz), but the most extreme cases suffered from higher levels of drift. The extent of drift varied across scanners which both linear and nonlinear drifts were observed., Competing Interests: Declaration of Competing Interest Jack J. Miller would like to acknowledge the support of a Novo Nordisk Research Fellowship run in conjunction with the University of Oxford. Francisco Reyes-Madrigal has served as a speaker for Janssen (Johnson & Johnson) and AstraZeneca. Marc Thioux and Pim van Dijk were supported by The Netherlands Organization for Health Research and Development (ZonMW) and the Dorhout Mees Foundation. All other authors have no conflict of interest to declare., (Copyright © 2021. Published by Elsevier Inc.)

The optic radiations (OR) are white matter tracts forming the posterior part of the visual ways. As an important inter-individual variability exists, atlases may be inefficient to locate the OR in a given subject. We designed a fully automatic method to delimitate the OR on a magnetic resonance imaging using tractography. On 15 healthy subjects, we evaluated the validity of our method by comparing the outputs to the Jülich post-mortem histological atlas, and its reproducibility. We also evaluated its feasibility on 98 multiple sclerosis (MS) patients. We correlated different visual outcomes with the inflammatory lesions volume within the OR reconstructed with different methods (our method, atlas, TractSeg). Our method reconstructed the OR bundle in all healthy subjects (< 2 h for most of them), and was reproducible. It demonstrated good classification indexes: sensitivity up to 0.996, specificity up to 0.993, Dice coefficient up to 0.842, and an area under the receiver operating characteristic (ROC) curve of 0.981. Our method reconstructed the OR in 91 of the 98 MS patients (92.9%, < 6 h for most of patients). Compared to an atlas-based approach and the TractSeg method, the inflammatory lesions volume in the OR measured with our method better correlated with the visual cortex volume, visual acuity and mean peripapillar retinal nerve fiber layer thickness. Our method seems to be efficient to reconstruct the OR in healthy subjects, and seems applicable to MS patients. It may be more relevant than an atlas based approach.

Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C. elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C. elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The combination of near-infrared (NIR) and visible wavelengths in light microscopy for biological studies is increasingly common. For example, many fields of biology are developing the use of NIR for optogenetics, in which an NIR laser induces a change in gene expression and/or protein function. One major technical barrier in working with both NIR and visible light on an optical microscope is obtaining their precise coalignment at the imaging plane position. Photon upconverting particles (UCPs) can bridge this gap as they are excited by NIR light but emit in the visible range via an anti-Stokes luminescence mechanism. Here, two different UCPs have been identified, high-efficiency micro 540 -UCPs and lower efficiency nano 545 -UCPs, that respond to NIR light and emit visible light with high photostability even at very high NIR power densities (>25 000 Suns). Both of these UCPs can be rapidly and reversibly excited by visible and NIR light and emit light at visible wavelengths detectable with standard emission settings used for Green Fluorescent Protein (GFP), a commonly used genetically encoded fluorophore. However, the high efficiency micro 540 -UCPs were suboptimal for NIR and visible light coalignment, due to their larger size and spatial broadening from particle-to-particle energy transfer consistent with a long-lived excited state and saturated power dependence. In contrast, the lower efficiency nano-UCPs were superior for precise coalignment of the NIR beam with the visible light path (∼2 μm versus ∼8 μm beam broadening, respectively) consistent with limited particle-to-particle energy transfer, superlinear power dependence for emission, and much smaller particle size. Furthermore, the nano-UCPs were superior to a traditional two-camera method for NIR and visible light path alignment in an in vivo Infrared-Laser-Evoked Gene Operator (IR-LEGO) optogenetics assay in the budding yeast Saccharomyces cerevisiae. In summary, nano-UCPs are powerful new tools for coaligning NIR and visible light paths on a light microscope.

Oocytes are extremely large cells that have to coordinate accurate chromosome segregation, asymmetric cytoplasm partitioning together with their own development as fertilizable gametes. For this, they undergo both global (cell cycle progression related) and local changes. It is therefore essential to be able to monitor local changes as they take place in live maturing oocytes. We describe here a method to follow RanGTP gradients using FRET technology in vivo.

C. elegans is a powerful metazoan model system to address fundamental questions in cell and developmental biology. Research in C. elegans has traditionally focused on genetic, physiological, and cell biological approaches. However, C. elegans is also a facile system for biochemistry: worms are easy to grow in large quantities, the functionality of tagged fusion proteins can be assessed using mutants or RNAi, and the relevance of putative interaction partners can be rapidly tested in vivo. Combining biochemistry with function-based genetic and RNA interference screens can rapidly accelerate the delineation of protein networks and pathways in diverse contexts. In this chapter, we focus on two strategies to identify protein-protein interactions: single-step immunoprecipitation and tandem affinity purification. We describe methods for growth of worms in large-scale liquid culture, preparation of worm and embryo extracts, immunoprecipitation, and tandem affinity purification. In addition, we describe methods to test specificity of antibodies, strategies for optimizing starting material, and approaches to distinguish specific from non-specific interactions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin beta. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.