Metabolic RNA labeling for probing RNA dynamics in bacteria.

Metabolic labeling of RNAs with noncanonical nucleosides that are chemically active, followed by chemoselective conjugation with imaging probes or enrichment tags, has emerged as a powerful method for studying RNA transcription and degradation in eukaryotes. However, metabolic RNA labeling is not applicable for prokaryotes, in which the complexity and distinctness of gene regulation largely remain to be explored. Here, we report 2'-deoxy-2'-azidoguanosine (AzG) as a noncanonical nucleoside compatible with metabolic labeling of bacterial RNAs. With AzG, we develop AIR-seq (azidonucleoside-incorporated RNA sequencing), which enables genome-wide analysis of transcription upon heat stress in Escherichia coli. Furthermore, AIR-seq coupled with pulse-chase labeling allows for global analysis of bacterial RNA degradation. Finally, we demonstrate that RNAs of mouse gut microbiotas can be metabolically labeled with AzG in living animals. The AzG-enabled metabolic RNA labeling should find broad applications in studying RNA biology in various bacterial species.

Optical Cell Tagging for Spatially Resolved Single-Cell RNA Sequencing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for profiling gene expression of distinct cell populations at the single-cell level. However, the information of the positions of cells within the multicellular samples is missing in scRNA-seq datasets. To overcome this limitation, we herein develop OpTAG (optical cell tagging) as a new chemical platform for attaching functional tags onto cell surfaces in a spatially resolved manner. With OpTAG, we establish OpTAG-seq, which enables spatially resolved scRNA-seq. We apply OpTAG-seq to investigate the spatially defined transcriptional program in migrating cancer cells and identified a list of genes that are potential regulators for cancer cell migration and invasion. OpTAG-seq provides a convenient method for mapping cellular heterogeneity with spatial information within multicellular biological systems.

Chemical Tagging of Protein Lipoylation.

Protein lipoylation is a post-translational modification of emerging importance in both prokaryotes and eukaryotes. However, labeling and large-scale profiling of protein lipoylation remain challenging. Here, we report the development of iLCL (iodoacetamide-assisted lipoate-cyclooctyne ligation), a chemoselective reaction that enables chemical tagging of protein lipoylation. We demonstrate that the cyclic disulfide of lipoamide but not linear disulfides can selectively react with iodoacetamide to produce sulfenic acid, which can be conjugated with cyclooctyne probes. iLCL enables tagging of lipoylated proteins for gel-based detection and cellular imaging. Furthermore, we apply iLCL for proteomic profiling of lipoylated proteins in both bacteria and mammalian cells. In addition to all of the eight known lipoylated proteins, we identified seven candidates for novel lipoylated proteins. The iLCL strategy should facilitate uncovering the biological function of protein lipoylation.

Synchronously sexual maturity in hermaphrodite fish as revealed by transcriptome analysis in Plectropomus leopardus.

Leopard coral grouper (Plectropomus leopardus) is a type of hermaphrodite fish, but the mechanisms of gonadal development and gametogenesis remain unclear. In the present study, we performed histological observation and transcriptomic analysis during the process of sexual differentiation in P. leopardus. According to the histological results, sexual differentiation was completed at 15 months old, developed synchronously in male and female individuals at 2 years old, and matured synchronously at 3 years old. Comparative transcriptomic analyses showed that the gonadal had differentiated by 15 months old, with enrichment of pathways associated with cell proliferation, transcriptional metabolism, and germline stem cell differentiation. Furthermore, cilium movement and fatty acid anabolism, which are associated with spermatogenesis and oocyte growth, were significantly enriched at 3 years old. In addition, key genes associated with male and female sex differentiation, such as amh, dmrt1, dmrt2a, zp4, sox3, gdf9, and gsdf, were identified by weighted gene co-expression network analysis (WGCNA). Finally, the localization and expression of the key genes amh and sox3 were observed in different cell types within the testes and ovaries, reflecting the development of the testes and ovaries, respectively. All the evidence indicates that P. leopardus is a hermaphrodite and synchronously sexually mature fish. Our study complements the gonadal development patterns of hermaphroditic fish by providing new insights into the molecular mechanisms underlying sexual differentiation and sex change in hermaphroditic groupers.

Cellular-scale proximity labeling for recording cell spatial organization in mouse tissues.

Proximity labeling has emerged as a powerful strategy for interrogating cell-cell interactions. However, the nanometer-scale labeling radius impedes the use of current methods for indirect cell communications and makes recording cell spatial organization in tissue samples difficult. Here, we develop quinone methide-assisted identification of cell spatial organization (QMID), a chemical strategy with the labeling radius matching the cell dimension. The activating enzyme is installed on the surface of bait cells, which produces QM electrophiles that can diffuse across micrometers and label proximal prey cells independent of cell-cell contacts. In cell coculture, QMID reveals gene expression of macrophages that are regulated by spatial proximity to tumor cells. Furthermore, QMID enables labeling and isolation of proximal cells of CD4+ and CD8+ T cells in the mouse spleen, and subsequent single-cell RNA sequencing uncovers distinctive cell populations and gene expression patterns within the immune niches of specific T cell subtypes. QMID should facilitate dissecting cell spatial organization in various tissues.

Genome-wide association study reveals genomic loci of sex differentiation and gonadal development in Plectropomus leopardus.

Introduction: Plectropomus leopardus, a commercially significant marine fish, is primarily found in the Western Pacific regions and along the coast of Southeast Asia. A thorough analysis of the molecular mechanisms involved in sex differentiation is crucial for gaining a comprehensive understanding of gonadal development and improving sex control breeding. However, the relevant fundamental studies of P. leopardus are relatively lacking. Methods: In this study, a genome-wide association study (GWAS) was conducted to investigate the genetic basis mechanism of sex differentiation and gonadal developmental traits in P. leopardus utilizing about 6,850,000 high-quality single-nucleotide polymorphisms (SNPs) derived from 168 individuals (including 126 females and 42 males) by the genome-wide efficient mixed-model association (GEMMA) algorithm. Results: The results of these single-trait GWASs showed that 46 SNP loci (-log10 p > 7) significantly associated with sex differentiation, and gonadal development traits were distributed in multiple different chromosomes, which suggested the analyzed traits were all complex traits under multi-locus control. A total of 1,838 potential candidate genes were obtained by considering a less-stringent threshold (-log10 p > 6) and ±100 kb regions surrounding the significant genomic loci. Moreover, 31 candidate genes were identified through a comprehensive analysis of significant GWAS peaks, gene ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, including taf7, ddx6, apoeb, sgk1, a2m, usf1, hsd3b7, dll4, xbp1, tet3, esr1, and gli3. These trait-associated genes have been shown to be involved in germline development, male sex differentiation, gonad morphogenesis, hormone receptor binding, oocyte development, male gonad development, steroidogenesis, estrogen-synthetic pathway, etc. Discussion: In the present study, multiple genomic loci of P. leopardus associated with sex differentiation and gonadal development traits were identified for the first time by using GWAS, providing a valuable resource for further research on the molecular genetic mechanism and sex control in P. leopardus. Our results also can contribute to understanding the genetic basis of the sex differentiation mechanism and gonadal development process in grouper fish.

Strength-limiting damage and its mitigation in CAD-CAM zirconia-reinforced lithium-silicate ceramics machined in a fully crystallized state.

OBJECTIVES: The objective was to explore how clinically relevant machining process and heat treatment influence damage accumulation and strength degradation in lithium silicate-based glass ceramics machined in the fully crystallized state. METHODS: A commercial zirconia-reinforced lithium silicate (ZLS) glass ceramic with a fully developed microstructure (Celtra® Duo) was studied. Disk-shaped specimens (nominal 10 mm diameter and 1 mm thickness) were fabricated either using a CAD-CAM process, creating a clinically relevant dental restoration surface, or were sectioned from water-jet cut cylindrical blocks with their critical surfaces consistently polished. Bi-axial flexure strength (BFS) was determined in a ball-on-ring configuration, and fractographic analysis was performed on failed specimens. XRD, AFM and SEM measurements were conducted before and after heat treatment. For each sample group, BFS was correlated with surface roughness. A two-way ANOVA and post-hoc Tukey tests were used to determine differences in BFS between machining and heat treatment groups (ɑ = 0.05). RESULTS: A two-way ANOVA demonstrated that BFS was influenced by fabrication route (p < 0.01) with CAD-CAM specimens exhibiting significantly lower mean BFS. A factorial interaction was observed between heat treatment and machining route (p < 0.01), where a significant strengthening effect of post-manufacture heat treatment was noted for CAD-CAM specimens but not sectioned and polished samples. CAD-CAM specimens exhibited sub-surface lateral cracks alongside radial cracks near fracture origin which were not observed for polished specimens. BFS did not correlate with surface roughness for polished specimens, and no change in microstructure was detectable by XRD following heat treatment. SIGNIFICANCE: The mechanical properties of the ZLS ceramic material studied were highly sensitive to the initial surface defect integral associated with manufacturing route and order of operations. CAD-CAM manufacturing procedures result in significant strength-limiting damage which is likely to influence restoration performance; however, this can be partially mitigated by post-machining heat treatment.

Photo-polymerisation variables influence the structure and subsequent thermal response of dental resin matrices.

OBJECTIVE: The structure of the polymer phase of dental resin-based-composites is highly sensitive to photo-polymerisation variables. The objective of this study was to understand how different polymer structures, generated with different photo-polymerisation protocols, respond to thermal perturbation. METHODS: Experimental resins were prepared from a series of Bis-GMA/TEGDMA blends (40/60, 50/50 and 60/40 wt.%), with either Camphorquinone/DMAEMA or Lucirin TPO as the photo-initiator system. Resins were photo-polymerised, in a disc geometry, at either relatively 'high' (3000 mW cm-2 for 6 s) or 'low' (300 mW cm-2 for 60 s) irradiances ensuring matched radiant exposures (18 J cm-2). Specimens were heated, from 20-160 °C at a rate of 5 °C min-1, whilst simultaneous synchrotron X-ray scattering measurements were taken at 5 °C increments to determine changes in polymer chain segment extension and medium-range order as a function of temperature. For each unique resin composition (n = 3), differential scanning calorimetry was used to measure glass transition temperatures using the same heating protocol. A paired t-test was used to determine significant differences in the glass transition temperature between irradiance protocols and photo-initiator chemistry at ɑ = 0.05. RESULTS: Resins pre-polymerised through the use of TPO and or high irradiances demonstrated a reduced rate of chain extension indicative of lower thermal expansion and a larger decrease in relative order when heated below the glass transition temperature. Above the transition temperature, differences in the rate of chain extension were negligible, but slower converted systems showed greater relative order. There was no significant difference in the glass transition temperature between different photo-initiator systems or irradiance protocols. SIGNIFICANCE: The evolution of chain extension and medium-range order during heating is dependent on the initial polymer structure which is influenced by photo-polymerisation variables. Less ordered systems, generated at faster rates of reactive group conversion displayed reduced chain extension below the glass transition temperature and maintained lower order throughout heating.

Origin of micro-scale heterogeneity in polymerisation of photo-activated resin composites.

Photo-activated resin composites are widely used in industry and medicine. Despite extensive chemical characterisation, the micro-scale pattern of resin matrix reactive group conversion between filler particles is not fully understood. Using an advanced synchrotron-based wide-field IR imaging system and state-of-the-art Mie scattering corrections, we observe how the presence of monodispersed silica filler particles in a methacrylate based resin reduces local conversion and chemical bond strain in the polymer phase. Here we show that heterogeneity originates from a lower converted and reduced bond strain boundary layer encapsulating each particle, whilst at larger inter-particulate distances light attenuation and monomer mobility predominantly influence conversion. Increased conversion corresponds to greater bond strain, however, strain generation appears sensitive to differences in conversion rate and implies subtle distinctions in the final polymer structure. We expect these findings to inform current predictive models of mechanical behaviour in polymer-composite materials, particularly at the resin-filler interface.

Extracellular vesicles derived from ODN-stimulated macrophages transfer and activate Cdc42 in recipient cells and thereby increase cellular permissiveness to EV uptake.

Endosomal Toll-like receptors (TLRs) mediate intracellular innate immunity via the recognition of DNA and RNA sequences. Recent work has reported a role for extracellular vesicles (EVs), known to transfer various nucleic acids, in uptake of TLR-activating molecules, raising speculation about possible roles of EVs in innate immune surveillance. Whether EV-mediated uptake is a general mechanism, however, was unresolved; and the molecular machinery that might be involved was unknown. We show that, when macrophages are stimulated with the TLR9 agonist CpG oligodeoxynucleotides (ODN), the secreted EVs transport ODN into naïve macrophages and induce the release of chemokine TNF-α. In addition, these EVs transfer Cdc42 into recipient cells, resulting in further enhancement of their cellular uptake. Transport of ODN and Cdc42 from TLR9-activated macrophages to naïve cells via EVs exerts synergetic effects in propagation of the intracellular immune response, suggesting a general mechanism of EV-mediated uptake of pathogen-associated molecular patterns.

Strength-limiting damage in lithium silicate glass-ceramics associated with CAD-CAM.

OBJECTIVE: The fabrication of all-ceramic restorations using Computer Aided Design and Computer Aided Manufacturing (CAD-CAM) most commonly involves subtractive machining which results in strength-limiting, surface and sub-surface damage in the resultant prosthesis. The objective was to explore how clinically relevant machining-process variables, and material variables, affect damage accumulation in lithium silicate glass-ceramics. METHODS: Three commercial lithium silicate glass-ceramics (IPS e.max® CAD, Celtra® Duo and Vita Suprinity®) were selected. For each material, two groups of disk-shaped specimens were fabricated (n=15), using a CAD-CAM process, creating surfaces equivalent to those generated for a dental restoration, or alternatively, using a highly controlled laboratory process generating disk-shaped test specimens with a consistent polished surface. Bi-axial flexure strength (BFS) was determined in a ball-on-ring configuration and fractographic analyses performed. For each material BFS was correlated with machining sequence and with surface roughness. RESULTS: BFS was significantly influenced by material substrate (p<0.01) and by fabrication route (p<0.01). A significant factorial interaction (p<0.01) identified that the magnitude of changes in BFS when comparing the two specimen fabrication routes, was dependent on substrate type. The polished control specimens exhibited a significantly increased BFS when compared with the CAD-CAM counterparts for all materials. IPS e.max® CAD and Celtra® Duo showed a 44 and 46% reduction in mean BFS for the CAD-CAM specimens when compared with the polished counterparts, respectively. In contrast, Vita Suprinity® showed the least disparity in mean BFS (21%) but the greatest variance in BFS data. SIGNIFICANCE: All CAD-CAM specimens showed evidence of machining introduced damage in the form of median and radial cracks at sites either coincident with, or peripheral to the failure origin. Subtractive machining introduced significant strength limiting damage that is not eliminated by heat treatments applied for either microstructure development (IPS e.max® CAD and Vita Suprinity®) or annealing/crack blunting (Celtra® Duo).

Multishape and Temperature Memory Effects by Strong Physical Confinement in Poly(propylene carbonate)/Graphene Oxide Nanocomposites.

The importance of filler-matrix interactions is generally recognized for mechanical property enhancement; their direct impact by physical confinement on diverse functional properties has remained poorly explored. We report here our effort in achieving versatile shape memory performances for a biodegradable poly(propylene carbonate) (PPC) matrix containing high contents of graphene oxide (GO). The excellent dispersion in the entire filler range (up to 20 wt %) allows precise morphological tuning, along with physical filler-matrix interactions, contributing overall to a strong nanoconfinement effect that positively affects the thermomechanical properties of nanocomposites. Only one glass-transition temperature (Tg) of PPC is detected when the GO content is below 10 wt %, corresponding to a slightly confined system, whereas two distinct Tg's are observed with a GO content over 10 wt %, corresponding to a highly confined system. As such, a tunable multishape memory effect can be achieved simply by tuning the filler contents. A dual-shape memory effect (DSME) is observed for a slightly confined system, whereas a triple-shape memory effect (TSME) can be achieved by deformation at two distinct Tg's for a highly confined system. More importantly, it is interesting to find that the switch temperature (Tsw) evolves linearly with the programing temperature (Tprog) for both slightly and highly confined systems, with Tsw ≈ Tprog for a highly confined system but Tsw < Tprog for a slightly confined system. Our work suggests a highly flexible approach to take advantage of the strong nanoconfinement effect by tuning the content of GO within a single polymer to access versatile SMEs, such as DSME and TSME, and the temperature memory effect.