Computational optical sectioning having an incoherent multiscale spreading style pertaining to lightfield microscopy

Chronic inflammatory diseases like rheumatoid arthritis are characterized by a deficit in fully functional regulatory T cells. DNA-methylation inhibitors have previously been shown to promote regulatory T cell responses and, in the present study, we evaluated their potential to ameliorate chronic and acute animal models of rheumatoid arthritis. Of the drugs tested, decitabine was the most effective, producing a sustained therapeutic effect that was dependent on indoleamine 2,3-dioxygenase (IDO) and was associated with expansion of induced regulatory T cells, particularly at the site of disease activity. Treatment with decitabine also caused apoptosis of Th1 and Th17 cells in active arthritis in a highly selective manner. The molecular basis for this selectivity was shown to be ENT1, a nucleoside transporter, which facilitates intracellular entry of the drug and is up-regulated on effector T cells during active arthritis. ZLEHDFMK It was further shown that short-term treatment with decitabine resulted in the generation of a population of regulatory T cells that were able to suppress arthritis upon adoptive transfer. In summary, a therapeutic approach using an approved drug is described that treats active inflammatory disease effectively and generates robust regulatory T cells with the IDO-dependent capacity to maintain remission.Tissue-on-chip systems represent promising platforms for monitoring and controlling tissue functions in vitro for various purposes in biomedical research. The two-dimensional (2D) layouts of these constructs constrain the types of interactions that can be studied and limit their relevance to three-dimensional (3D) tissues. The development of 3D electronic scaffolds and microphysiological devices with geometries and functions tailored to realistic 3D tissues has the potential to create important possibilities in advanced sensing and control. This study presents classes of compliant 3D frameworks that incorporate microscale strain sensors for high-sensitivity measurements of contractile forces of engineered optogenetic muscle tissue rings, supported by quantitative simulations. Compared with traditional approaches based on optical microscopy, these 3D mechanical frameworks and sensing systems can measure not only motions but also contractile forces with high accuracy and high temporal resolution. Results of active tension force measurements of engineered muscle rings under different stimulation conditions in long-term monitoring settings for over 5 wk and in response to various chemical and drug doses demonstrate the utility of such platforms in sensing and modulation of muscle and other tissues. Possibilities for applications range from drug screening and disease modeling to biohybrid robotic engineering.Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for the distinct functions of Smc5/6 is poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 holo-complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that the Smc5/6 complex possesses several unique features, while sharing some architectural characteristics with other SMC complexes. In contrast to arm-folded structures of cohesin and condensin, Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5/Nse6 subcomplex, with the latter also serving as a linchpin connecting distal parts of the complex. Our 3.0-Å resolution cryoelectron microscopy structure of the Nse5/Nse6 core further reveals a clasped-hand topology and a dimeric interface important for cell growth. Finally, we provide evidence that Nse5/Nse6 uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative study identifies distinct structural features of the Smc5/6 complex and functional cooperation among its coevolved unique subunits.Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. However, plants usually house multiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma. Plants are known to sanction strains that do not fix nitrogen, but nonfixers are rare in field settings, while intermediate fixers are common. Here, we modeled how plants should respond to an intermediate fixer that was otherwise isogenic and tested model predictions using pea plants. Intermediate fixers were only tolerated when a better strain was not available. In agreement with model predictions, nodules containing the intermediate-fixing strain were large and healthy when the only alternative was a nonfixer, but nodules of the intermediate-fixing strain were small and white when the plant was coinoculated with a more effective strain. The reduction in nodule size was preceded by a lower carbon supply to the nodule even before differences in nodule size could be observed. Sanctioned nodules had reduced rates of nitrogen fixation, and in later developmental stages, sanctioned nodules contained fewer viable bacteria than nonsanctioned nodules. This indicates that legumes can make conditional decisions, most likely by comparing a local nodule-dependent cue of nitrogen output with a global cue, giving them remarkable control over their symbiotic partners.Tsunami generation from earthquake-induced seafloor deformations has long been recognized as a major hazard to coastal areas. Strike-slip faulting has generally been considered insufficient for triggering large tsunamis, except through the generation of submarine landslides. Herein, we demonstrate that ground motions due to strike-slip earthquakes can contribute to the generation of large tsunamis (>1 m), under rather generic conditions. To this end, we developed a computational framework that integrates models for earthquake rupture dynamics with models of tsunami generation and propagation. The three-dimensional time-dependent vertical and horizontal ground motions from spontaneous dynamic rupture models are used to drive boundary motions in the tsunami model. Our results suggest that supershear ruptures propagating along strike-slip faults, traversing narrow and shallow bays, are prime candidates for tsunami generation. We show that dynamic focusing and the large horizontal displacements, characteristic of strike-slip earthquakes on long faults, are critical drivers for the tsunami hazard. These findings point to intrinsic mechanisms for sizable tsunami generation by strike-slip faulting, which do not require complex seismic sources, landslides, or complicated bathymetry. Furthermore, our model identifies three distinct phases in the tsunamic motion, an instantaneous dynamic phase, a lagging coseismic phase, and a postseismic phase, each of which may affect coastal areas differently. We conclude that near-source tsunami hazards and risk from strike-slip faulting need to be re-evaluated.Details of the fine structure of a particular transcript may be assessed by S1 mapping or ribonuclease protection. The use of either of these techniques allows the detection of the 5' and 3' ends of a particular mRNA, as well as the splice junctions, precursors, and processing intermediates of mRNA. Primer extension provides a measure of the amount of a particular mRNA species and allows an exact determination of the 5' end of the mRNA. These three methods are introduced here.Escape behaviors, orienting reflexes, and social behaviors in Xenopus laevis tadpoles have been well-documented in the literature. Schooling behavior experiments allow for the observation of tadpole social interactions and in the past have been used to characterize behavioral deficits in models of neurodevelopmental disorders. Unlike other species of frogs, Xenopus tadpoles show polarized schooling. Not only do tadpoles aggregate, they also swim in the same direction. Quantifying both aggregation and relative swim angle can give us an important measure of social behavior and sensory integration. Past iterations of these experiments have required the continued presence of an experimenter throughout the duration of each trial and relied on expensive software for subsequent data analysis. The instrument configuration and analysis protocol outlined here provide an automated method to assess schooling by delivering a series of timed vibratory stimuli to a group of tadpoles to induce swimming behavior and then controlling a camera to document their positions via still images. Both stimulus delivery and image acquisition are automated using the Python programming language. Analysis is done using ImageJ and custom Python scripts, which are provided in this protocol. The specific equipment configuration and scripts shown here provide one solution, but other equipment and custom scripts can be substituted.For mapping the 5' termini of mRNA molecules, primer extension is the method of choice. A purified oligonucleotide is end-labeled using polynucleotide kinase. The probe and a population of mRNA are allowed to hybridize, and the primers and template are used to carry out reverse transcription using an enzyme cloned from the Moloney murine leukemia virus. The primer extension products are separated on a denaturing polyacrylamide gel and analyzed by radiography.In this protocol a randomly labeled single-stranded RNA probe is prepared and then hybridized to a population of mRNA molecules. The RNAs are digested with a mixture of RNase A and RNase T1. link2 The hybrid molecules, which are resistant to the RNases, are separated and analyzed using gel electrophoresis and radiography.This protocol provides details for nuclease S1 mapping of mRNA using a uniformly labeled, single-stranded DNA probe. DNA-RNA hybrids are generated, which are subsequently digested with nuclease S1. The digestion products are separated using gel electrophoresis and analyzed by radiography.Genetic immunization has been useful in vaccine technology and can also be used to generate immune responses to novel proteins. It is particularly useful when a properly folded protein is difficult to isolate or make in recombinant form and the required antibody must recognize a conformational epitope. Injecting cDNA allows the protein to be expressed in native form by the animal's own cells in vivo and then presented to the immune system. link3 Genetic immunization is most effective for the generation of antibodies if the cDNA encodes for secreted or cell-surface proteins to make them accessible to the immune system. It also has been successful in generating antibody responses to difficult protein targets such as G-coupled protein receptors, ion channel proteins, and other multiple membrane-spanning proteins. High-affinity antibodies tend to be favored because the proteins are expressed at low levels and are constantly present for presentation to the immune system. In addition, several months can be saved obtaining sufficient amounts of properly folded, soluble recombinant protein or high-expressing transfected cells.