Single-Cell RNA-Seq Mapping of Human Thymopoiesis Reveals Lineage Specification Trajectories and a Commitment Spectrum in T Cell Development.

The challenges in recapitulating in vivo human T cell development in laboratory models have posed a barrier to understanding human thymopoiesis. Here, we used single-cell RNA sequencing (sRNA-seq) to interrogate the rare CD34+ progenitor and the more differentiated CD34- fractions in the human postnatal thymus. CD34+ thymic progenitors were comprised of a spectrum of specification and commitment states characterized by multilineage priming followed by gradual T cell commitment. The earliest progenitors in the differentiation trajectory were CD7- and expressed a stem-cell-like transcriptional profile, but had also initiated T cell priming. Clustering analysis identified a CD34+ subpopulation primed for the plasmacytoid dendritic lineage, suggesting an intrathymic dendritic specification pathway. CD2 expression defined T cell commitment stages where loss of B cell potential preceded that of myeloid potential. These datasets delineate gene expression profiles spanning key differentiation events in human thymopoiesis and provide a resource for the further study of human T cell development.

Interfacial engineering of plasmonic nanoparticle metasurfaces.

SignificanceMolecules interacting with metallic nanostructures can show tunable exciton-plasmon coupling, ranging from weak to strong. One factor that influences the interactions is the spatial organization of the molecules relative to the localized plasmon-enhanced electromagnetic fields. In this work, we show that the arrangement of aromatic dye molecules can be tuned within plasmonic hotspots by interfacial engineering of nanoparticle surfaces. By controlling the local chemical and physical interactions, we could modulate lasing thresholds. Surface-functionalized plasmonic metasurfaces open prospects for programmable light-matter interactions at the nanoscale.

Open Cross-gap Gold Nanocubes with Strong, Large-Area, Symmetric Electromagnetic Field Enhancement for On-Particle Molecular-Fingerprint Raman Bioassays.

Plasmonic nanoparticles with an externally open nanogap can localize the electromagnetic (EM) field inside the gap and directly detect the target via the open nanogap with surface-enhanced Raman scattering (SERS). It would be beneficial to design and synthesize the open gap nanoprobes in a high yield for obtaining uniform and quantitative signals from randomly oriented nanoparticles and utilizing these particles for direct SERS analysis. Here, we report a facile strategy to synthesize open cross-gap (X-gap) nanocubes (OXNCs) with size- and EM field-tunable gaps in a high yield. The site-specific growth of Au budding structures at the corners of the AuNC using the principle that the Au deposition rate is faster than the surface diffusion rate of the adatoms allows for a uniform X-gap formation. The average SERS enhancement factor (EF) for the OXNCs with 2.6 nm X-gaps was 1.2 × 109, and the EFs were narrowly distributed within 1 order of magnitude for ∼93% of the measured OXNCs. OXNCs consistently displayed strong EM field enhancement on large particle surfaces for widely varying incident light polarization directions, and this can be attributed to the symmetric X-gap geometry and the availability of these gaps on all 6 faces of a cube. Finally, the OXNC probes with varying X-gap sizes have been utilized in directly detecting biomolecules with varying sizes without Raman dyes. The concept, synthetic method, and biosensing results shown here with OXNCs pave the way for designing, synthesizing, and utilizing plasmonic nanoparticles for selective, quantitative molecular-fingerprint Raman sensing and imaging applications.

The State of Use and Utility of Negative Controls in Pharmacoepidemiologic Studies.

Uses of real-world data in drug safety and effectiveness studies are often challenged by various sources of bias. We undertook a systematic search of the published literature through September 2020 to evaluate the state of use and utility of negative controls to address bias in pharmacoepidemiologic studies. Two reviewers independently evaluated study eligibility and abstracted data. Our search identified 184 eligible studies for inclusion. Cohort studies (115, 63%) and administrative data (114, 62%) were, respectively, the most common study design and data type used. Most studies used negative control outcomes (91, 50%), and for most studies the target source of bias was unmeasured confounding (93, 51%). We identified 4 utility domains of negative controls: 1) bias detection (149, 81%), 2) bias correction (16, 9%), 3) P-value calibration (8, 4%), and 4) performance assessment of different methods used in drug safety studies (31, 17%). The most popular methodologies used were the 95% confidence interval and P-value calibration. In addition, we identified 2 reference sets with structured steps to check the causality assumption of the negative control. While negative controls are powerful tools in bias detection, we found many studies lacked checking the underlying assumptions. This article is part of a Special Collection on Pharmacoepidemiology.

Synergistic Inclusion Effects of Hard Magnetic Nanorods on the Magnetomechanical Actuation of Soft Magnetic Microsphere-Based Polymer Composites.

The magnetomechanical actuation of micropillars is developed for the contactless manipulation of miniaturized actuators and microtextured surfaces. Anisotropic geometry of micropillars can significantly enhance the magnetic actuation compared with their isotropic counterparts by directional stress distributions. However, this strategy is not viable for triangular micropillars owing to insufficient anisotropy. In this study, a significant improvement in the magnetic actuation of triangular micropillars using composite magnetic particles is reported. A minute and optimal amount of hard magnetic gamma-ferrite nanorods are hybridized with soft magnetic iron microspheres to generate synergistic effects of magnetic coupling and percolation phenomenon on the magnetic actuation of polymer composites. The addition of 1 wt% face-centered cubic-phased gamma-ferrite nanorods suppresses the magnetic coupling interference of body-centered cubic-phased iron microspheres. Furthermore, the nanorods reduce the percolation threshold by participating in the percolation of the microspheres. A systematic compositional study on the magnetization and magnetorheological properties reveals that the coupling effect dominates the percolation effect at a low magnetic field, whereas the percolation effect governs the magnetic actuation at a high magnetic field. This hybrid approach can help in designing material constituents for effective magnetic actuators and robotic systems that can sensitively respond to an external magnetic field.

Light-Matter Interactions in Hybrid Material Metasurfaces.

This Review focuses on the integration of plasmonic and dielectric metasurfaces with emissive or stimuli-responsive materials for manipulating light-matter interactions at the nanoscale. Metasurfaces, engineered planar structures with rationally designed building blocks, can change the local phase and intensity of electromagnetic waves at the subwavelength unit level and offers more degrees of freedom to control the flow of light. A combination of metasurfaces and nanoscale emitters facilitates access to weak and strong coupling regimes for enhanced photoluminescence, nanoscale lasing, controlled quantum emission, and formation of exciton-polaritons. In addition to emissive materials, functional materials that respond to external stimuli can be combined with metasurfaces to engineer tunable nanophotonic devices. Emerging metasurface designs including surface-functionalized, chemically tunable, and multilayer hybrid metasurfaces open prospects for diverse applications, including photocatalysis, sensing, displays, and quantum information.

Efficient Labeling of Vesicles with Lipophilic Fluorescent Dyes via the Salt-Change Method.

Fluorescent labeling allows for imaging and tracking of vesicles down to single-particle level. Among several options to introduce fluorescence, staining of lipid membranes with lipophilic dyes provides a straightforward approach without interfering with vesicle content. However, incorporating lipophilic molecules into vesicle membranes in an aqueous solution is generally not efficient because of their low water solubility. Here, we describe a simple, fast (<30 min), and highly effective procedure for fluorescent labeling of vesicles including natural extracellular vesicles. By adjusting the ionic strength of the staining buffer with NaCl, the aggregation status of DiI, a representative lipophilic tracer, can be controlled reversibly. Using cell-derived vesicles as a model system, we show that dispersion of DiI under low-salt condition improved its incorporation into vesicles by a factor of 290. In addition, increasing NaCl concentration after labeling induced free dye molecules to form aggregates, which can be filtered and thus effectively removed without ultracentrifugation. We consistently observed 6- to 85-fold increases in the labeled vesicle count across different types of dyes and vesicles. The method is expected to reduce the concern about off-target labeling resulting from the use of high concentrations of dyes.

Ultranarrow plasmon resonances from annealed nanoparticle lattices.

This paper reports how the spectral linewidths of plasmon resonances can be narrowed down to a few nanometers by optimizing the morphology, surface roughness, and crystallinity of metal nanoparticles (NPs) in two-dimensional (2D) lattices. We developed thermal annealing procedures to achieve ultranarrow surface lattice resonances (SLRs) with full-width at half-maxima linewidths as narrow as 4 nm from arrays of Au, Ag, Al, and Cu NPs. Besides annealing, we developed a chemical vapor deposition process to use Cu NPs as catalytic substrates for graphene growth. Graphene-encapsulated Cu NPs showed the narrowest SLR linewidths (2 nm) and were stable for months. These ultranarrow SLR nanocavity modes supported even narrower lasing emission spectra and high nonlinearity in the input-output light-light curves.

Admixture of divergent genomes facilitates hybridization across species in the family Brassicaceae.

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus. We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration. Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis-elements of duplicated genes are associated with convergent expression. Genome-wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa-originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation. Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis-trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.

Nontrivial, Unconventional Electrochromic Behaviors of Plasmonic Nanocubes.

Plasmonic electrochromism, a change in the localized surface plasmon resonance (LSPR) with an applied electric potential, has been attracting increasing attention for the development of spectroscopic tools or optoelectronic systems. There is a consensus on the mechanism of plasmonic electrochromism based on the classical capacitor and the Drude model. However, the electrochromic behaviors of metallic nanoparticles in narrow optical windows have been demonstrated only with small monotonic LSPR shifts, which limits the use of the electrochromism. Here, we observed three distinct electrochromic behaviors of gold nanocubes with a wide potential range through in situ dark-field electrospectroscopy. Interestingly, the nanocubes show a faster frequency shift under the highly negative potential, and this opens the possibility of largely tunable electrochromic LSPR shifts. The reversibility of the electrochemical switching with these cubes are also shown. We attribute this unexpected change beyond classical understandings to the material-specific quantum mechanical electronic structures of the plasmonic materials.

Strong Coupling Between Plasmons and Molecular Excitons in Metal-Organic Frameworks.

This Letter describes strong coupling of densely packed molecular emitters in metal-organic frameworks (MOFs) and plasmonic nanoparticle (NP) lattices. Porphyrin-derived ligands with small transition dipole moments in an ordered MOF film were grown on Ag NP arrays. Angle-resolved optical measurements of the MOF-NP lattice system showed the formation of a polariton that is lower in energy and does not cross the uncoupled MOF Q1 band. Modeling predicted the upper polariton energy and a calculated Rabi splitting of 110 meV. The coupling strength was systematically controlled by detuning the plasmon energy by changing the refractive index of the solvents infiltrating the MOF pores. Through transient absorption spectroscopy, we found that the lower polariton decays quickly at shorter time scales (<500 ps) and slowly at longer times because of energy transfer from the upper polariton. This hybrid system demonstrates how MOFs can function as an accessible excitonic material for polariton chemistry.

Alteration of payload in extracellular vesicles by crosstalk with mesenchymal stem cells from different origin.

BACKGROUND: The application of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) requires customized materials to target disease or cell damage. We hypothesized that EVs exert different inflammatory effects on one recipient cell, although stem cells of different origins in humans have similar payloads. RESULTS: Here, the payload of EVs released by crosstalk between MSCs and human middle ear epithelial cells (HMEECs) extracted from adipose tissue, bone marrow and tonsils significantly increased the level of anti-inflammatory factors. EVs derived from the co-culture medium decreased TNF-α, COX-2, IL-1ß, and IL-6 levels to approximately zero within 3 h in HMEECs. Expression of miR-638 and amyloid-ß A4 precursor protein-binding family A member 2 was analyzed using microarrays and gene ontology analysis, respectively. CONCLUSIONS: In conclusion, stem cells of different origins have different payloads through crosstalk with recipient-specific cells. Inducing specific factors in EVs by co-culture with MSCs could be valuable in regenerative medicine.

Protective effect of MSC-derived exosomes against cisplatin-induced apoptosis via heat shock protein 70 in auditory explant model.

Therapeutics based on stem cell technology, including stem cell-derived exosomes, have emerged in recent years for the treatment of what were otherwise considered incurable diseases. In this study, we evaluated the efficacy of human MSC-derived exosomes for protection against cisplatin induced ototoxic hearing loss. Incubation of cochlear explants with MSC-derived exosomes prior to addition of cisplatin induced a reduction in cisplatin-induced drug toxicity in auditory hair cells but not when the exosomes were introduced simultaneously with or after cisplatin. The delivery of MSC-derived exosomes to cochlear explants was confirmed by the increasing protein levels of the exosome markers CD63 and HSP70 to reduce apoptosis. These results were consistent with those from a model in which MSC-derived exosomes protect auditory hair cells from cisplatin-induced drug toxicity in an ex vivo cochlear explant model and support future studies into the therapeutic benefits of stem cell-derived exosomes in clinical applications.

Revealing biomass heterosis in the allodiploid xBrassicoraphanus, a hybrid between Brassica rapa and Raphanus sativus, through integrated transcriptome and metabolites analysis.

BACKGROUND: Heterosis is biologically important but the molecular basis of the phenomenon is poorly understood. We characterized intergeneric hybrids between B. rapa cv. Chiifu and R. sativus cv. WK10039 as an extreme example of heterosis. Taking advantage of clear heterosis phenotypes and the genetic distance between parents, we performed transcriptome and metabolite analysis to decipher the molecular basis of heterosis. RESULTS: The heterosis was expressed as fresh weight in the field and as inflorescence stem length in the glass house. Flowering time, distributed as a normal segregating population, ranged from the early flowering of one parent to the late flowering of the other, in contrast to the homogeneous flowering time in a typical F1 population, indicating unstable allelic interactions. The transcriptome and metabolome both indicated that sugar metabolism was altered, suggesting that the change in metabolism was linked to the heterosis. Because alleles were not shared between the hybridized genomes, classic models only partly explain this heterosis, indicating that other mechanisms are involved. CONCLUSION: The differential expression of genes for primary and secondary metabolism, along with the altered metabolite profiles, suggests that heterosis could involve a change in balance between primary and secondary metabolism.

Enhancement of Magneto-Mechanical Actuation of Micropillar Arrays by Anisotropic Stress Distribution.

Magnetically active shape-reconfigurable microarrays undergo programmed actuation according to the arrangement of magnetic dipoles within the structures, achieving complex twisting and bending deformations. Cylindrical micropillars have been widely used to date, whose circular cross-sections lead to identical actuation regardless of the actuating direction. In this study, micropillars with triangular or rectangular cross-sections are designed and fabricated to introduce preferential actuation directions and explore the limits of their actuation. Using such structures, controlled liquid wetting is demonstrated on micropillar surfaces. Liquid droplets pinned on magnetic micropillar arrays undergo directional spreading when the pillars are actuated as depinning of the droplets is enabled only in certain directions. The enhanced deformation due to direction dependent magneto-mechanical actuation suggests that micropillar arrays can be fundamentally tailored to possess application specific responses and opens up opportunities to exploit more complex designs such as micropillars with polygonal cross sections. Such tunable wetting of liquids on microarray surfaces has potential to improve printing technologies via contactless reconfiguration of stamp geometry by magnetic field manipulation.

Deficiency of rice hexokinase HXK5 impairs synthesis and utilization of starch in pollen grains and causes male sterility.

There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

Improvement of stem cell-derived exosome release efficiency by surface-modified nanoparticles.

BACKGROUND: Mesenchymal stem cells (MSCs) are pluripotent stromal cells that release extracellular vesicles (EVs). EVs contain various growth factors and antioxidants that can positively affect the surrounding cells. Nanoscale MSC-derived EVs, such as exosomes, have been developed as bio-stable nano-type materials. However, some issues, such as low yield and difficulty in quantification, limit their use. We hypothesized that enhancing exosome production using nanoparticles would stimulate the release of intracellular molecules. RESULTS: The aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified, positively charged nanoparticles and exosome generation from MSCs. We determined that Rab7, a late endosome and auto-phagosome marker, was increased upon exosome expression and was associated with autophagosome formation. CONCLUSIONS: It was concluded that the nanoparticles we developed were transported to the lysosome by clathrin-mediated endocytosis. additionally, entered nanoparticles stimulated that autophagy related factors to release exosome from the MSC. MSC-derived exosomes using nanoparticles may increase exosome yield and enable the discovery of nanoparticle-induced genetic factors.

Induced Short-Term Hearing Loss due to Stimulation of Age-Related Factors by Intermittent Hypoxia, High-Fat Diet, and Galactose Injection.

Age-related hearing loss (ARHL) is the most common sensory disorder among the elderly, associated with aging and auditory hair cell death due to oxidative-stress-induced mitochondrial dysfunction. Although transgenic mice and long-term aging induction cultures have been used to study ARHL, there are currently no ARHL animal models that can be stimulated by intermittent environmental changes. In this study, an ARHL animal model was established by inducing continuous oxidative stress to promote short-term aging of cells, determined on the basis of expression of hearing-loss-induced phenotypes and aging-related factors. The incidence of hearing loss was significantly higher in dual- and triple-exposure conditions than in intermittent hypoxic conditions, high-fat diet (HFD), or d-galactose injection alone. Continuous oxidative stress and HFD accelerated cellular aging. An increase in Ucp2, usually expressed during mitochondrial dysfunction, was observed. Expression of Cdh23, Slc26a4, Kcnq4, Myo7a, and Myo6, which are ARHL-related factors, were modified by oxidative stress in the cells of the hearing organ. We found that intermittent hypoxia, HFD, and galactose injection accelerated cellular aging in the short term. Thus, we anticipate that the development of this hearing loss animal model, which reflects the effects of intermittent environmental changes, will benefit future research on ARHL.

Verification of the Necessity of the Tolyl Group of PF-543 for Sphingosine Kinase 1 Inhibitory Activity.

PF-543, the most potent sphingosine kinase (SK) inhibitor, does not demonstrate effective anticancer activity in some cancer cells, unlike other known SK1 inhibitors. PF-543 has a non-lipid structure with a unique toluene backbone; however, the importance of this structure remains unclear. Therefore, the purpose of this study was to investigate changes in SK inhibitory and anticancer activities and to explore the role of the tolyl group structure of PF-543 through various modifications. We transformed the methyl group of PF-543 into hydrogen, fluorine, and hydroxy. PF-543 derivatives in which the methyl group was substituted by hydrogen and fluorine (compound 5) demonstrated SK1 inhibitory and anticancer activities similar to PF-543. Moreover, we performed molecular modeling studies of PF-543 and compound 5. To assess the metabolic stability of PF-543 and compound 5, we determined their degree of degradation using the liver microsomes of four different animal species (human, dog, rat, and mouse). However, both PF-543 and compound 5 showed poor microsomal stability. Therefore, for the medical applications of PF-543, the structural modifications of its other parts may be necessary. Our results provide important information for the design of additional PF-543 analogs.

Precisely Shaped, Uniformly Formed Gold Nanocubes with Ultrahigh Reproducibility in Single-Particle Scattering and Surface-Enhanced Raman Scattering.

Synthesizing plasmonic nanostructures in an ultraprecise manner is of paramount importance because the nanometer-scale structural details can significantly affect their plasmonic properties. Au nanocubes (AuNCs) have been a highly promising, heavily studied nanostructure with high potential in various fields, but an ultraprecise synthesis from 10 to 100 nm in size over a large number of AuNCs has not been well established. Precisely structured AuNC-based studies for a highly reproducible, quantitative plasmonic signal generation [e.g., quantitative surface-enhanced Raman scattering (SERS)] are needed for reliable use and exploration in the beneficial properties of AuNCs. Here, we developed a strategy for AuNC synthesis with the desired size and shape, ranging from 17 to 78 nm particularly with highly controlled corner sharpness, by precisely controlling the growth rate of different facets and AuNC-specific flocculation which enabled ultrahigh yields (∼98-99%). Importantly, the precisely shaped AuNCs can scatter light in a spectrally reproducible manner, and the SERS enhancement factors (EFs) for the AuNC dimers are very narrowly distributed (the EFs of 72 nm sharp-cornered cube dimers have a distribution within 1 order of magnitude). Our results pave the paths to ultrahigh yield synthesis of metal nanocubes with a precise size and shape and offer single-particle-level spectral controllability and reproducibility over a large number of particles.