Oxford Nanopore Technologies' instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of ...significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing.
We have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries' average read length over manual slow pipetting.
SNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.
The charged particle community is looking for techniques exploiting proton interactions instead of X-ray absorption for creating images of human tissue. Due to multiple Coulomb scattering inside the ...measured object it has shown to be highly non-trivial to achieve sufficient spatial resolution. We present imaging of biological tissue with a proton microscope. This device relies on magnetic optics, distinguishing it from most published proton imaging methods. For these methods reducing the data acquisition time to a clinically acceptable level has turned out to be challenging. In a proton microscope, data acquisition and processing are much simpler. This device even allows imaging in real time. The primary medical application will be image guidance in proton radiosurgery. Proton images demonstrating the potential for this application are presented. Tomographic reconstructions are included to raise awareness of the possibility of high-resolution proton tomography using magneto-optics.
Despite well-established protocols for cardiopulmonary resuscitation training, performance during real-life cardiac arrests can be suboptimal. Understanding personal characteristics which could ...influenceperformance of high-quality chest compressions could provide insight into the practice-performance gap. This study examined chest compressionperformance, while employing feedback and introducing code team sounds as an anxiety-inducing factor in registered nurses using a cardiopulmonary resuscitation training manikin.
Participants included 120 registered nurses with basic life support certification randomized to one of the following groups: no feedback and no code team sounds, feedback without code team sounds, or feedback with code team sounds. Chest compression sessions occurred at baseline, 30-days and 60-days. Demographic variables and anxiety level were also collected. The primary outcome was chest compression performance, defined as average percent of time with correct rate and percent with correct depth as captured by the defibrillator. Statistical analysis included linear mixed effects analysis.
The effect of feedback on chest compression performance depended on the value of other parameters. The benefit of feedback on the primary outcome depended on: age, with feedback less beneficial among older participants (p = 0.0413); and time, with feedback more beneficial with repetition (p = 0.011). These interactions also affected the outcome percent of time with correct compression depth. Increased anxiety was associated with decreased percent correct compression depth (p < 0.001).
Feedback emerged as important in determining chest compression performance. Chest compression quality was limited by the performer’s age and anxiety level. Future research should focus on identifying factors related to individual characteristics which may influence chest compression performance.
Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) ...on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01+ and KIR3DL05+ macaques with and without defined ligands for these receptors with SIVmac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16+ population during acute infection and sustained increases in the CD16+ and CD16-CD56- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFα) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFα upregulation by KIR3DL01+, but not by KIR3DL05+ NK cells. Significant increases in the frequency of KIR3DL01+ (but not KIR3DL05+) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56+ and CD16-CD56- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01+ NK cells in tissues that support high levels of virus replication.
Purpose:
The use of motion mitigation techniques such as tracking and gating in particle therapy requires real-time knowledge of tumor position with millimeter precision. The aim of this ...phantom-based study was to evaluate the option of diagnostic ultrasound (US) imaging (sonography) as real-time motion detection method for scanned heavy ion beam irradiation of moving targets.
Methods:
For this pilot experiment, a tumor surrogate was moved inside a water bath along two-dimensional trajectories. A rubber ball was used for this purpose. This ball was moved by a robotic arm in two dimensions lateral to the heavy ion beam. Trajectories having a period of 3 s and peak to peak amplitude of 20 mm were used. Square radiation fields of
\documentclass12pt{minimal}\begin{document}$3 \times 3\; \textrm {cm}^2$\end{document}
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were irradiated on radiosensitive films with a 200 MeV/u beam of calcium ions having a FWHM of 6 mm. Pencil beam scanning and beam tracking were employed. The films were attached on the robotic arm and thus moved with the rubber ball. The position of the rubber ball was continuously measured by a US tracking system (Mediri GmbH, Heidelberg) and sent to the GSI therapy control system (TCS). This position was used as tracking vector. Position reconstruction from the US tracking system and data communication introduced a delay leading to a position error of several millimeters. An artificial neural network (ANN) was implemented in the TCS to predict motion from US measurements and thus to compensate for the delay.
Results:
Using ANN delay compensation and large motion amplitudes, the authors could produce irradiation patterns with a few percent inhomogeneity and about 1 mm geometrical conformity.
Conclusions:
This pilot experiment suggests that diagnostic US should be further investigated as dose-free, high frame-rate, and model-independent motion detection method for scanning heavy ion beam irradiation of moving targets.
Abstract Modern techniques as ion beam therapy or 4D imaging require precise target position information. However, target motion particularly in the abdomen due to respiration or patient movement is ...still a challenge and demands methods that detect and compensate this motion. Ultrasound represents a non-invasive, dose-free and model-independent alternative to fluoroscopy, respiration belt or optical tracking of the patient surface. Thus, ultrasound based motion tracking was integrated into irradiation with actively scanned heavy ions. In a first in vitro experiment, the ultrasound tracking system was used to compensate diverse sinusoidal target motions in two dimensions. A time delay of ∼200 ms between target motion and reported position data was compensated by a prediction algorithm (artificial neural network). The irradiated films proved feasibility of the proposed method. Furthermore, a practicable and reliable calibration workflow was developed to enable the transformation of ultrasound tracking data to the coordinates of the treatment delivery or imaging system – even if the ultrasound probe moves due to respiration. A first proof of principle experiment was performed during time-resolved positron emission tomography (4DPET) to test the calibration workflow and to show the accuracy of an ultrasound based motion tracking in vitro . The results showed that optical ultrasound tracking can reach acceptable accuracies and encourage further research.
Macaques provide the most widely used nonhuman primate models for studying the immunology and pathogenesis of human diseases. Although the macaque major histocompatibility complex (MHC) region shares ...most features with the human leukocyte antigen (HLA) region, macaques have an expanded repertoire of MHC class I genes. Although a chimera of two rhesus macaque MHC haplotypes was first published in 2004, the structural diversity of MHC genomic organization in macaques remains poorly understood owing to a lack of adequate genomic reference sequences. We used ultralong Oxford Nanopore and high-accuracy Pacific Biosciences (PacBio) HiFi sequences to fully assemble the ∼5.2-Mb M3 haplotype of an MHC-homozygous, Mauritian-origin cynomolgus macaque (
). The MHC homozygosity allowed us to assemble a single MHC haplotype unambiguously and avoid chimeric assemblies that hampered previous efforts to characterize this exceptionally complex genomic region in macaques. The high quality of this new assembly is exemplified by the identification of an extended cluster of six
genes that contains a recent duplication with a highly similar ∼48.5-kb block of sequence. The MHC class II region of this M3 haplotype is similar to the previously sequenced rhesus macaque haplotype and HLA class II haplotypes. The MHC class I region, in contrast, contains 13
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genes, four
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genes, and three
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genes (vs. 19
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, two
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, and one
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in the previously sequenced haplotype). These results provide an unambiguously assembled single contiguous cynomolgus macaque MHC haplotype with fully curated gene annotations that will inform infectious disease and transplantation research.
Mauritian-origin cynomolgus macaques (MCMs) serve as a powerful nonhuman primate model in biomedical research due to their unique genetic homogeneity, which simplifies experimental designs. Despite ...their extensive use, a comprehensive understanding of crucial immune-regulating gene families, particularly killer Ig-like receptors (KIR) and NK group 2 (NKG2), has been hindered by the lack of detailed genomic reference assemblies. In this study, we employ advanced long-read sequencing techniques to completely assemble eight KIR and seven NKG2 genomic haplotypes, providing an extensive insight into the structural and allelic diversity of these immunoregulatory gene clusters. Leveraging these genomic resources, we prototype a strategy for genotyping KIR and NKG2 using short-read, whole-exome capture data, illustrating the potential for cost-effective multilocus genotyping at colony scale. These results mark a significant enhancement for biomedical research in MCMs and underscore the feasibility of broad-scale genetic investigations.
Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining ...transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute "pre-peak" phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque 'elite controllers' with a short-course of dextran sulfate sodium (DSS)-stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host's capacity to contain infection.
The Karlsruhe Tritium Neutrino (KATRIN) experiment will determine the mass of the electron neutrino with a sensitivity of 0.2 eV (90% CL) via a measurement of the β-spectrum of gaseous tritium near ...its endpoint of E0 = 18.57 keV. An ultra-low background of about b = 10 mHz is among the requirements on reaching this sensitivity. In the KATRIN main beam line, two spectrometers of MAC-E filter type are used in tandem configuration. This setup, however, produces a Penning trap, which could lead to increased background. We have performed test measurements showing that the filter energy of the pre-spectrometer can be reduced by several keV in order to diminish this trap. These measurements were analyzed with the help of a complex computer simulation, modeling multiple electron reflections from both the detector and the photoelectric electron source used in our test setup.
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