CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration.

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

Reorganization of sensory networks after subcortical vestibular infarcts: A longitudinal symptom-related voxel-based morphometry study.

BACKGROUND AND PURPOSE: We aimed to delineate common principles of reorganization after infarcts of the subcortical vestibular circuitry related to the clinical symptomatology. Our hypothesis was that the recovery of specific symptoms is associated with changes in distinct regions within the core vestibular, somatosensory, and visual cortical and subcortical networks. METHODS: We used voxel- and surface-based morphometry to investigate structural reorganization of subcortical and cortical brain areas in 42 patients with a unilateral, subcortical infarct with vestibular and ocular motor deficits in the acute phase. The patients received structural neuroimaging and clinical monitoring twice (acute phase and after 6 months) to detect within-subject changes over time. RESULTS: In patients with vestibular signs such as tilts of the subjective visual vertical (SVV) and ocular torsion in the acute phase, significant volumetric increases in the superficial white matter around the parieto-opercular (retro-)insular vestibular cortex (PIVC) were found at follow-up. In patients with SVV tilts, spontaneous nystagmus, and rotatory vertigo in the acute phase, gray matter volume decreases were located in the cerebellum and the visual cortex bilaterally at follow-up. Patients with saccade pathology demonstrated volumetric decreases in cerebellar, thalamic, and cortical centers for ocular motor control. CONCLUSIONS: The findings support the role of the PIVC as the key hub for vestibular processing and reorganization. The volumetric decreases represent the reciprocal interaction of the vestibular, visual, and ocular motor systems during self-location and egomotion detection. A modulation in vestibular and ocular motor as well as visual networks was induced independently of the vestibular lesion site.

High-molecular-weight organic matter in the particles of comet 67P/Churyumov-Gerasimenko.

The presence of solid carbonaceous matter in cometary dust was established by the detection of elements such as carbon, hydrogen, oxygen and nitrogen in particles from comet 1P/Halley. Such matter is generally thought to have originated in the interstellar medium, but it might have formed in the solar nebula-the cloud of gas and dust that was left over after the Sun formed. This solid carbonaceous material cannot be observed from Earth, so it has eluded unambiguous characterization. Many gaseous organic molecules, however, have been observed; they come mostly from the sublimation of ices at the surface or in the subsurface of cometary nuclei. These ices could have been formed from material inherited from the interstellar medium that suffered little processing in the solar nebula. Here we report the in situ detection of solid organic matter in the dust particles emitted by comet 67P/Churyumov-Gerasimenko; the carbon in this organic material is bound in very large macromolecular compounds, analogous to the insoluble organic matter found in the carbonaceous chondrite meteorites. The organic matter in meteorites might have formed in the interstellar medium and/or the solar nebula, but was almost certainly modified in the meteorites' parent bodies. We conclude that the observed cometary carbonaceous solid matter could have the same origin as the meteoritic insoluble organic matter, but suffered less modification before and/or after being incorporated into the comet.

Potassic, high-silica Hadean crust.

Understanding Hadean (>4 Ga) Earth requires knowledge of its crust. The composition of the crust and volatiles migrating through it directly influence the makeup of the atmosphere, the composition of seawater, and nutrient availability. Despite its importance, there is little known and less agreed upon regarding the nature of the Hadean crust. By analyzing the 87Sr/86Sr ratio of apatite inclusions in Archean zircons from Nuvvuagittuq, Canada, we show that its protolith had formed a high (>1) Rb/Sr ratio reservoir by at least 4.2 Ga. This result implies that the early crust had a broad range of igneous rocks, extending from mafic to highly silicic compositions.

Human Exploration of Enclosed Spaces through Echolocation.

Some blind humans have developed echolocation, as a method of navigation in space. Echolocation is a truly active sense because subjects analyze echoes of dedicated, self-generated sounds to assess space around them. Using a special virtual space technique, we assess how humans perceive enclosed spaces through echolocation, thereby revealing the interplay between sensory and vocal-motor neural activity while humans perform this task. Sighted subjects were trained to detect small changes in virtual-room size analyzing real-time generated echoes of their vocalizations. Individual differences in performance were related to the type and number of vocalizations produced. We then asked subjects to estimate virtual-room size with either active or passive sounds while measuring their brain activity with fMRI. Subjects were better at estimating room size when actively vocalizing. This was reflected in the hemodynamic activity of vocal-motor cortices, even after individual motor and sensory components were removed. Activity in these areas also varied with perceived room size, although the vocal-motor output was unchanged. In addition, thalamic and auditory-midbrain activity was correlated with perceived room size; a likely result of top-down auditory pathways for human echolocation, comparable with those described in echolocating bats. Our data provide evidence that human echolocation is supported by active sensing, both behaviorally and in terms of brain activity. The neural sensory-motor coupling complements the fundamental acoustic motor-sensory coupling via the environment in echolocation.SIGNIFICANCE STATEMENT Passive listening is the predominant method for examining brain activity during echolocation, the auditory analysis of self-generated sounds. We show that sighted humans perform better when they actively vocalize than during passive listening. Correspondingly, vocal motor and cerebellar activity is greater during active echolocation than vocalization alone. Motor and subcortical auditory brain activity covaries with the auditory percept, although motor output is unchanged. Our results reveal behaviorally relevant neural sensory-motor coupling during echolocation.

Multisensory vestibular, vestibular-auditory, and auditory network effects revealed by parametric sound pressure stimulation.

Multisensory convergence and sensorimotor integration are important aspects for the mediation of higher vestibular cognitive functions at the cortical level. In contrast to the integration of vestibulo-visual or vestibulo-tactile perception, much less is known about the neural mechanism that mediates the integration of vestibular-otolith (linear acceleration/translation/gravity detection) and auditory processing. Vestibular-otolith and auditory afferents can be simultaneously activated using loud sound pressure stimulation, which is routinely used for testing cervical and ocular vestibular evoked myogenic potentials (VEMPs) in clinical neurotological testing. Due to the simultaneous activation of afferents there is always an auditory confound problem in fMRI studies of the neural topology of these systems. Here, we demonstrate that the auditory confounding problem can be overcome in a novel way that does not require the assumption of simple subtraction and additionally allows detection of non-linear changes in the response due to vestibular-otolith interference. We used a parametric sound pressure stimulation design that took each subject's vestibular stimulation threshold into account and analyzed for changes in BOLD-response below and above vestibular-otolith threshold. This approach helped to investigate the functional neuroanatomy of sound-induced auditory and vestibular integration using functional magnetic resonance imaging (fMRI). Results revealed that auditory and vestibular convergence are contained in overlapping regions of the caudal part of the superior temporal gyrus (STG) and the posterior insula. In addition, there are regions that were responsive only to suprathreshold stimulations, suggesting vestibular (otolith) signal processing in these areas. Based on these parametric analyses, we suggest that the caudal part of the STG and posterior insula could contain areas of vestibular contribution to auditory processing, i.e., higher vestibular cortices that provide multisensory integration that is important for tasks such as spatial localization of sound.

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation.

UNLABELLED: Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging. IMPORTANCE: The essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

Magnetic vestibular stimulation modulates default mode network fluctuations.

Strong magnetic fields (>1 Tesla) can cause dizziness and it was recently shown that healthy subjects (resting in total darkness) developed a persistent nystagmus even when remaining completely motionless within a MR tomograph. Consequently, it was speculated that this magnetic vestibular stimulation (MVS) might influence fMRI results, as nystagmus is indicative of an imbalance in the vestibular system, potentially influencing other systems via multisensory vestibular interactions. The objective of our study was to investigate whether MVS does indeed modulate BOLD signal fluctuations. We recorded eye movements, as well as, resting-state fMRI of 30 volunteers in darkness at 1.5 T and 3.0 T to answer the question whether MVS modulated parts of the default mode resting-state network (DMN) in accordance with the Lorentz-force model for MVS, while distinguishing this from the known signal increase due to field strength related imaging effects. Our results showed that modulation of the default mode network occurred mainly in areas associated with vestibular and ocular motor function, and was in accordance with the Lorentz-force model, i.e., double than the expected signal scaling due to field strength alone. We discuss the implications of our findings for the interpretation of studies using resting-state fMRI, especially those concerning vestibular research. We conclude that MVS needs to be considered in vestibular research to avoid biased results, but it might also offer the possibility of manipulating network dynamics and may thus help in studying the brain as a dynamical system.

Management of an Implant-Supported Fixed Partial Denture in the Esthetic Zone in a Patient With a Very Limited Mouth Opening: A Case Report.

In the maxillary anterior area, the esthetic integration of prosthetic restorations is a challenge, particularly for screw-retained implant prostheses. This case report presents the management and clinical outcome of an old partial edentulous maxillary jaw in an esthetic zone in a young patient with a very limited mouth opening. This patient was rehabilitated with an implant screw-retained fixed partial denture (FPD) using both digital and conventional techniques.

Portable, single nucleotide polymorphism-specific duplex assay for virus surveillance in wastewater.

The Argonaute protein from the archaeon Pyrococcus furiosus (PfAgo) is a DNA-guided nuclease that targets DNA with any sequence. We designed a virus detection assay in which the PfAgo enzyme cleaves the reporter probe, thus generating fluorescent signals when amplicons from a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay contain target sequences. We confirmed that the RT-LAMP-PfAgo assay for the SARS-CoV-2 Delta variant produced significantly higher fluorescent signals (p < 0.001) when a single nucleotide polymorphism (SNP), exclusive to the Delta variant, was present, compared to the samples without the SNP. Additionally, the duplex assay for Pepper mild mottle virus (PMMOV) and SARS-CoV-2 detection produced specific fluorescent signals (FAM or ROX) only when the corresponding sequences were present. Furthermore, the RT-LAMP-PfAgo assay does not require dilution to reduce the impact of environmental inhibitors. The limit of detection of the PMMOV assay, determined with 30 wastewater samples, was 28 gc/µL, with a 95 % confidence interval of [11,103]. Finally, using a point-of-use device, the RT-LAMP-PfAgo assay successfully detected PMMOV in wastewater samples. Based on our findings, we conclude that the RT-LAMP-PfAgo assay can be used as a portable, SNP-specific duplex assay, which will significantly improve virus surveillance in wastewater.

Maximum forces applied to the orbital floor after fractures.

The objective of this study was to measure the force on and displacement of completely detached intraorbital tissue from the bony orbit, as a worst-case scenario after orbital trauma, to preserve the maximum load and predict the necessary strength of reconstruction materials. Six fresh-frozen human heads were used, and orbital floor defects in the right and left orbits were created by the direct impact of 3.0 J onto the globe and infraorbital rim. The orbital floor defect sizes and displacements were evaluated after performing a Le Fort I osteotomy. In addition, after the repositioning of the completely detached intraorbital tissue, the forces and displacements were measured. The mean orbital floor defect sizes were 208.3 (SD, 33.4) mm2 for globe impacts and 221.8 (SD, 53.1) mm2 for infraorbital impacts. The mean intraorbital tissue displacement after the impact and before repositioning was 5.6 (SD, 1.0) mm for globe impacts and 2.8 (SD, 0.7) mm for infraorbital impacts. After repositioning, the displacements were 0.8 (SD, 0.5) mm and 1.1 (SD, 0.7) mm, respectively. The measured forces were 0.10519 (SD, 0.00958) N without the incorporation and approximately 0.11128 (SD, 0.003599) N with the incorporation of reconstruction materials. The maximum forces on the completely detached orbital tissue were minimal (∼0.11 N) and suggest the use of collagen membranes as reconstruction materials for orbital floor defects, at least in medium-sized fractures.

Forces charging the orbital floor after orbital trauma.

The objectives of this study were (i) to evaluate different fracture mechanisms for orbital floor fractures and (ii) to measure forces and displacement of intraorbital tissue after orbital traumata to predict the necessity of strength for reconstruction materials. Six fresh frozen human heads were used, and orbital floor defects in the right and left orbit were created by a direct impact of 3.0 J onto the globe and infraorbital rim, respectively. Orbital floor defect sizes and displacement were evaluated after a Le Fort I osteotomy. In addition, after reposition of the intraorbital tissue, forces and displacement were measured. The orbital floor defect sizes were 208.3 (SD, 33.4) mm(2) for globe impact and 221.8 (SD, 53.1) mm(2) for infraorbital impact. The intraorbital tissue displacement after the impact and before reposition was 5.6 (SD, 1.0) mm for globe impact and 2.8 (SD, 0.7) mm for infraorbital impact. After reposition, the displacement was 0.8 (SD, 0.5) mm and 1.1 (SD, 0.7) mm, respectively. The measured applied forces were 0.061 (SD, 0.014) N for globe impact and 0.066 (SD, 0.022) N for infraorbital impact. Different fracture-inductive mechanisms are not reflected by the pattern of the fracture. The forces needed after reposition are minimal (~0.07 N), which may explain the success of PDS foils [poly-(p-dioxanone)] and collagen membranes as reconstruction materials.

White matter volume loss drives cortical reshaping after thalamic infarcts.

OBJECTIVE: The integration of somatosensory, ocular motor and vestibular signals is necessary for self-location in space and goal-directed action. We aimed to detect remote changes in the cerebral cortex after thalamic infarcts to reveal the thalamo-cortical connections necessary for multisensory processing and ocular motor control. METHODS: Thirteen patients with unilateral ischemic thalamic infarcts presenting with vestibular, somatosensory, and ocular motor symptoms were examined longitudinally in the acute phase and after six months. Voxel- and surface-based morphometry were used to detect changes in vestibular and multisensory cortical areas and known hubs of central ocular motor processing. The results were compared with functional connectivity data in 50 healthy volunteers. RESULTS: Patients with paramedian infarcts showed impaired saccades and vestibular perception, i.e., tilts of the subjective visual vertical (SVV). The most common complaint in these patients was double vision or vertigo / dizziness. Posterolateral thalamic infarcts led to tilts of the SVV and somatosensory deficits without vertigo. Tilts of the SVV were higher in paramedian compared to posterolateral infarcts (median 11.2° vs 3.8°). Vestibular and ocular motor symptoms recovered within six months. Somatosensory deficits persisted. Structural longitudinal imaging showed significant volume reduction in subcortical structures connected to the infarcted thalamic nuclei (vestibular nuclei region, dentate nucleus region, trigeminal root entry zone, medial lemniscus, superior colliculi). Volume loss was evident in connections to the frontal, parietal and cingulate lobes. Changes were larger in the ipsilesional hemisphere but were also detected in homotopical regions contralesionally. The white matter volume reduction led to deformation of the cortical projection zones of the infarcted nuclei. CONCLUSIONS: White matter volume loss after thalamic infarcts reflects sensory input from the brainstem as well the cortical projections of the main affected nuclei for sensory and ocular motor processing. Changes in the cortical geometry seem not to reflect gray matter atrophy but rather reshaping of the cortical surface due to the underlying white matter atrophy.

PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction.

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments. PlasmidMaker should greatly expand the potential of synthetic biology.

Structural and functional plasticity of the hippocampal formation in professional dancers and slackliners.

The acquisition of special skills can induce plastic changes in the human hippocampus, a finding demonstrated in expert navigators (Maguire et al. (2000) Proc Natl Acad Sci USA 97:4,398-403). Conversely, patients with acquired chronic bilateral vestibular loss develop atrophy of the hippocampus, which is associated with impaired spatial memory (Brandt et al. (2005) Brain 128:2,732-741). This suggests that spatial memory relies on vestibular input. In this study 21 professional dancers and slackliners were examined to assess whether balance training with extensive vestibulo-visual stimulation is associated with altered hippocampal formation volumes or spatial memory. Gray matter voxel-based morphometry showed smaller volumes in the anterior hippocampal formation and in parts of the parieto-insular vestibular cortex of the trained subjects but larger volumes in the posterior hippocampal formation and the lingual and fusiform gyri bilaterally. The local volumes in the right anterior hippocampal formation correlated negatively and those in the right posterior hippocampal formation positively with the amount of time spent training ballet/ice dancing or slacklining at the time of the study. There were no differences in general memory or in spatial memory as assessed by the virtual Morris water task. Trained subjects performed significantly better on a hippocampal formation-dependent task of nonspatial memory (transverse patterning). The smaller anterior hippocampal formation volumes of the trained subjects may be the result of a long-term suppression of destabilizing vestibular input. This is supported by the associated volume loss in the parieto-insular vestibular cortex. The larger volumes in the posterior hippocampal formation of the trained subjects might result from their increased utilization of visual cues for balance. This is supported by the concomitant larger volumes in visual areas like the lingual and fusiform gyri. Our findings indicate that there is a spatial separation of vestibular and visual processes in the human hippocampus.

Forces charging the orbital floor after fractures.

The objective of this study was first to establish a method to measure forces and displacement of the orbital content in defects of the orbital floor in truncated fresh and unfixed heads and second to characterize reconstruction materials with regard to punctuation strength and compression.Orbital floor defects (10 × 20 mm and 15 × 20 mm; 3 mm behind the orbital rim) were prepared after Le Fort I osteotomy. The values of force and displacement were recorded in 6 freshly frozen human heads. In addition, the punctuation strength of 2 reconstruction materials (polydioxanone [PDS] foil and collagen membrane) was evaluated using a Zwick Z010 TN1 universal testing machine. The forces of the orbital content (28.41 [SD, 1.6] g) applied to the defects of 10 × 20 mm and 15 × 20 mm with an intact periorbita were 0.04 (SD, 0.003) N (0.0002 MPa) and 0.07 (SD, 0.02) N (0.0002 MPa), respectively, and with a split periorbita were 0.06 (SD, 0.03) N (0.0003 MPa) and 0.08 (SD, 0.06) N (0.00026 MPa), respectively. The displacement values without reconstruction materials of the 10 × 20-mm and 15 × 20-mm defects were 0.94 (SD, 0.7) mm and 1.2 (SD, 0.5) mm, respectively. The PDS foil could withstand forces of 118.9 (SD, 14.1) N (0.375 MPa), and the collagen membrane could withstand forces of 44.5 (SD, 5.3) N (0.14 MPa). This is the first study to report forces charging the orbital floor. The presented results support the use of PDS foils and collagen membranes as reconstruction materials for orbital floor defects, at least in smaller and medium-sized fractures.

A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis.

The need for rapid, accurate, and scalable testing systems for COVID-19 diagnosis is clear and urgent. Here, we report a rapid Scalable and Portable Testing (SPOT) system consisting of a rapid, highly sensitive, and accurate assay and a battery-powered portable device for COVID-19 diagnosis. The SPOT assay comprises a one-pot reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) followed by PfAgo-based target sequence detection. It is capable of detecting the N gene and E gene in a multiplexed reaction with the limit of detection (LoD) of 0.44 copies/µL and 1.09 copies/µL, respectively, in SARS-CoV-2 virus-spiked saliva samples within 30 min. Moreover, the SPOT system is used to analyze 104 clinical saliva samples and identified 28/30 (93.3% sensitivity) SARS-CoV-2 positive samples (100% sensitivity if LoD is considered) and 73/74 (98.6% specificity) SARS-CoV-2 negative samples. This combination of speed, accuracy, sensitivity, and portability will enable high-volume, low-cost access to areas in need of urgent COVID-19 testing capabilities.

Cluster Analysis of Presolar Silicon Carbide Grains: Evaluation of Their Classification and Astrophysical Implications.

Cluster analysis of presolar silicon carbide grains based on literature data for 12C/13C, 14N/15N, δ 30Si/28Si, and δ 29Si/28Si including or not inferred initial 26Al/27Al data, reveals nine clusters agreeing with previously defined grain types but also highlighting new divisions. Mainstream grains reside in three clusters probably representing different parent star metallicities. One of these clusters has a compact core, with a narrow range of composition, pointing to an enhanced production of SiC grains in asymptotic giant branch (AGB) stars with a narrow range of masses and metallicities. The addition of 26Al/27Al data highlights a cluster of mainstream grains, enriched in 15N and 26Al, which cannot be explained by current AGB models. We defined two AB grain clusters, one with 15N and 26Al excesses, and the other with 14N and smaller 26Al excesses, in agreement with recent studies. Their definition does not use the solar N isotopic ratio as a divider, and the contour of the 26Al-rich AB cluster identified in this study is in better agreement with core-collapse supernova models. We also found a cluster with a mixture of putative nova and AB grains, which may have formed in supernova or nova environments. X grains make up two clusters, having either strongly correlated Si isotopic ratios or deviating from the 2/3 slope line in the Si 3-isotope plot. Finally, most Y and Z grains are jointly clustered, suggesting that the previous use of 12C/13C = 100 as a divider for Y grains was arbitrary. Our results show that cluster analysis is a powerful tool to interpret the data in light of stellar evolution and nucleosynthesis modeling and highlight the need of more multi-element isotopic data for better classification.

Structural reorganization of the cerebral cortex after vestibulo-cerebellar stroke.

OBJECTIVE: Structural reorganization following cerebellar infarcts is not yet known. This study aimed to demonstrate structural volumetric changes over time in the cortical vestibular and multisensory areas (i.e., brain plasticity) after acute cerebellar infarcts with vestibular and ocular motor symptoms. Additionally, we evaluated whether structural reorganization in the patients topographically correlates with cerebello-cortical connectivity that can be observed in healthy participants. METHODS: We obtained high-resolution structural imaging in seven patients with midline cerebellar infarcts at two time points. These data were compared to structural imaging of a group of healthy age-matched controls using voxel-based morphometry (2×2 ANOVA approach). The maximum overlap of the infarcts was used as a seed region for a separate resting-state functional connectivity analysis in healthy volunteers. RESULTS: Volumetric changes were detected in the multisensory cortical vestibular areas around the parieto-opercular and (retro-) insular cortex. Furthermore, structural reorganization was evident in parts of the frontal, temporal, parietal, limbic, and occipital lobes and reflected functional connections between the main infarct regions in the cerebellum and the cerebral cortex in healthy individuals. CONCLUSIONS: This study demonstrates structural reorganization in the parieto-opercular insular vestibular cortex after acute vestibulo-cerebellar infarcts. Additionally, the widely distributed structural reorganization after midline cerebellar infarcts provides additional in vivo evidence for the multifaceted contribution of cerebellar processing to cortical functions that extend beyond vestibular or ocular motor function.

TALEN outperforms Cas9 in editing heterochromatin target sites.

Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.