Heterogeneous plasma cells and long-lived subsets in response to immunization, autoantigen and microbiota.

Durable antibody immunity depends on long-lived plasma cells (LLPCs) that primarily reside in the bone marrow (BM). However, due to LLPC rarity, it has not been possible to define their phenotypes or determine their heterogeneity. By single-cell mRNA sequencing, cytometry and a genetic pulse-chase mouse model, we show that IgG and IgM LLPCs display an EpCAMhiCXCR3- phenotype, whereas IgA LLPCs are Ly6AhiTigit-. While IgG and IgA LLPCs are mainly contributed by somatically hypermutated cells following immunization or infection, cells with innate properties and public antibodies are found in IgA and IgM LLPC compartments. Particularly, IgM LLPCs are highly enriched with public clones shared among different individual animals, differentiated in a T cell-independent manner and have affinity for self-antigens and microbial-derived antigens. Taken together, our work reveals different routes toward LLPC development and paves the way for deeper understanding of cellular and molecular underpinnings of long-term antibody immunity.

Affinity-coupled CCL22 promotes positive selection in germinal centres.

Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (TFH) cells and, consequently, receive more contact-dependent T cell help1. As these GC B cells and TFH cells do not maintain long-lasting contacts in the chaotic GC environment2-4, it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on TFH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of TFH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from TFH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.

Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems.

Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.

A GPR174-CCL21 module imparts sexual dimorphism to humoral immunity.

Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males1-3. However, the mechanisms underlying such sexual dimorphism are not well understood. Here we show that there are intrinsic differences between the B cells that produce germinal centres in male and female mice. We find that antigen-activated male B cells do not position themselves as efficiently as female B cells in the centre of follicles in secondary lymphoid organs, in which germinal centres normally develop. Moreover, GPR174-an X-chromosome-encoded G-protein-coupled receptor-suppresses the formation of germinal centres in male, but not female, mice. This effect is intrinsic to B cells, and correlates with the GPR174-enhanced positioning of B cells towards the T-cell-B-cell border of follicles, and the distraction of male, but not female, B cells from S1PR2-driven follicle-centre localization. Biochemical fractionation of conditioned media that induce B-cell migration in a GPR174-dependent manner identifies CCL21 as a GPR174 ligand. In response to CCL21, GPR174 triggers a calcium flux and preferentially induces the migration of male B cells; GPR174 also becomes associated with more Gαi protein in male than in female B cells. Male B cells from orchidectomized mice exhibit impaired GPR174-mediated migration to CCL21, and testosterone treatment rescues this defect. Female B cells from testosterone-treated mice exhibit male-like GPR174-Gαi association and GPR174-mediated migration. Deleting GPR174 from male B cells causes more efficient positioning towards the follicular centre, the formation of more germinal centres and an increased susceptibility to B-cell-dependent experimental autoimmune encephalomyelitis. By identifying GPR174 as a receptor for CCL21 and demonstrating its sex-dependent control of B-cell positioning and participation in germinal centres, we have revealed a mechanism by which B-cell physiology is fine-tuned to impart sexual dimorphism to humoral immunity.

Enhancing Light Out-coupling in Perovskite Light-Emitting Diodes through Plasmonic Nanostructures.

Perovskite light-emitting diodes (PeLEDs) have emerged as promising candidates for lighting and display technologies owing to their high photoluminescence quantum efficiency and high carrier mobility. However, the performance of planar PeLEDs is limited by the out-coupling efficiency, predominantly governed by photonic losses at device interfaces. Most notably, the plasmonic loss at the metal electrode interfaces can account for up to 60% of the total loss. Here, we investigate the use of plasmonic nanostructures to improve the light out-coupling in PeLEDs. By integrating these nanostructures with PeLEDs, we have demonstrated an effectively reduced plasmonic loss and enhanced light out-coupling. As a result, the nanostructured PeLEDs exhibit an average 1.5-fold increase in external quantum efficiency and an ∼20-fold improvement in device lifetime. This finding offers a generic approach for enhancing light out-coupling, promising great potential to go beyond existing performance limitations.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

Functional analysis of the ocnE gene involved in nicotine-degradation pathways in Ochrobactrum intermedium SCUEC4 and its enzymatic properties.

The SCUEC4 strain of Ochrobactrum intermedium is a newly isolated bacterium that degrades nicotine can use nicotine as the sole carbon source via a series of enzymatic catalytic processes. The mechanisms underlying nicotine degradation in this bacterium and the corresponding functional genes remain unclear. Here, we analyzed the function and biological properties of the ocnE gene involved in the nicotine-degradation pathways in strain SCUEC4. The ocnE gene was cloned by PCR with total DNA of strain SCUEC4 and used to construct the recombinant plasmid pET28a-ocnE. The overexpression of the OcnE protein was detected by SDS-PAGE analysis, and study of the function of this protein was spectrophotometrically carried out by monitoring the changes of 2,5-dihydroxypyridine. Moreover, the effects of temperature, pH, and metal ions on the biological activities of the OcnE protein were analyzed. The optimal conditions for the biological activities of OcnE, a protein of approximately 37.6 kDa, were determined to be 25 °C, pH 7.0, and 25 µmol/L Fe2+, and the suitable storage conditions for the OcnE protein were 0 °C and pH 7.0. In conclusion, the ocnE gene is responsible for the ability of 2,5-dihydroxypyridine dioxygenase. These findings will be beneficial in clarifying the mechanisms of nicotine degradation in O. intermedium SCUEC4.

Improved Charge Transfer and Hot Spots by Doping and Modulating the Semiconductor Structure: A High Sensitivity and Renewability Surface-Enhanced Raman Spectroscopy Substrate.

Here, we develop a new method to improve the surface-enhanced Raman spectroscopy (SERS) activity of ZnO using Mg doping combined with noble metals. Highly aligned silver nanoparticles (AgNPs) decorated on an array of Mg-doped ZnO (MZO@Ag) were fabricated. Using rhodamine 6G as the probe molecule, SERS indicated that the MZO@Ag substrate possesses perfect sensitivity, homogeneity, and chemical stability. The enhancement mechanism of this substrate was analyzed in detail, and finite-difference time-domain (FDTD) simulations were used to examine "hot spot" distribution which generated gaps between the balls, the rods, and the stems. FDTD simulation calculated ( E/ E0)4 to be 2.5 × 106. Furthermore, the prepared substrates could degrade the target molecules in situ irradiated by visible light irradiation over the course of 40 min and then efficiently recover detectability through a recycling process. Our substrates were easy to fabricate, self-cleaning, and reusable. They are expected to provide new opportunities for the use of SERS in biological sensors, biomedical diagnostics, and food safety.

ZnO nanoparticles on MoS2 microflowers for ultrasensitive SERS detection of bisphenol A.

A heterojunction microcomposite was synthesized that consists of ZnO nanoparticles (ZnO NPs) anchored on MoS2 microflowers (MoS2 MFs). The material is shown to enable trace level detection of the pollutant bisphenol A (BPA). The microcomposite was characterized by XRD, XPS, SEM and TEM. In addition, coupling reaction between phenolic estrogens and Pauly's reagents was adopted to greatly enhance the SERS signal. BPA display a characteristic Raman band at 1592 cm-1 which can be used for its selective detection. The assay is highly sensitive and has a 1 nM detection limit which is the lowest among the reported semiconductor substrates. Graphical abstract MoS2/ZnO MCs SERS substrate broke through the application barrier of semiconductor composite materials in SERS substrates. It also sheds light on a deeper understanding of the charge-transfer based enhancement mechanism.

Thin-Film-Assisted Photothermal Deformation of Gold Nanoparticles: A Facile and In-Situ Strategy for Single-Plate-Based Devices.

Optical-induced shape transformation of single nanoparticles on substrates has shown benefits of simplicity and regularity for single-particle device fabrication and on-chip integration. However, most of the existing strategies are based on wet chemical growth and etching, which could lead to surface contamination with limited local selectivity and device compatibility. Shape deformation via the photothermal effect can overcome these issues but has limited versatility and tunability largely due to the high surface tension of the molten droplet. Here we show gold nanoparticles (Au NPs) can drastically transform into nanoplates under the irradiation of a continuous wave laser (446 nm). We reveal the dielectric thin film underneath the molten Au is critical in deforming the NP into faceted nanoplate under the drive of photothermophoretic forces, which is sufficient to counteract the surface tension of the molten droplet. Both experimental evidence and simulations support this thin-film-assisted photothermal deformation mechanism, which is local selective and generally applicable to differently shaped Au NPs. It provides a facile and robust strategy for single-plate-based device applications.

B cell-reactive triad of B cells, follicular helper and regulatory T cells at homeostasis.

Autoreactive B cells are silenced through receptor editing, clonal deletion and anergy induction. Additional autoreactive B cells are ignorant because of physical segregation from their cognate autoantigen. Unexpectedly, we find that follicular B cell-derived autoantigen, including cell surface molecules such as FcγRIIB, is a class of homeostatic autoantigen that can induce spontaneous germinal centers (GCs) and B cell-reactive autoantibodies in non-autoimmune animals with intact T and B cell repertoires. These B cell-reactive B cells form GCs in a manner dependent on spontaneous follicular helper T (TFH) cells, which preferentially recognize B cell-derived autoantigen, and in a manner constrained by spontaneous follicular regulatory T (TFR) cells, which also carry specificities for B cell-derived autoantigen. B cell-reactive GC cells are continuously generated and, following immunization or infection, become intermixed with foreign antigen-induced GCs. Production of plasma cells and antibodies derived from B cell-reactive GC cells are markedly enhanced by viral infection, potentially increasing the chance for autoimmunity. Consequently, immune homeostasis in healthy animals not only involves classical tolerance of silencing and ignoring autoreactive B cells but also entails a reactive equilibrium attained by a spontaneous B cell-reactive triad of B cells, TFH cells and TFR cells.

Optoelectronic tuning of plasmon resonances via optically modulated hot electrons.

Fast optical modulation of nanoplasmonics is fundamental for on-chip integration of all-optical devices. Although various strategies have been proposed for dynamic modulation of surface plasmons, critical issues of device compatibility and extremely low efficiency in the visible spectrum hamper the application of optoplasmonic nanochips. Here we establish an optoplasmonic system based on Au@Cu2-xS hybrid core-shell nanoparticles. The optical excitation of hot electrons and their charge transfer to the semiconductor coating (Cu2-xS) lead to lowered electron density of Au, which results in the red shift of the localized surface plasmon resonance. The hot electrons can also transport through the Cu2-xS layer to the metal substrate, which increases the conductance of the nanogap. As such, the coupled gap plasmon blue-shifts with a magnitude of up to ∼15 nm, depending on the excitation power and the thickness of the coatings, which agrees with numerical simulations. All of this optoelectronic tuning process is highly reversible, controllable and fast with a modulated laser beam, which is highly compatible and sufficiently useful for on-chip integration of nanophotonic devices.

Effect of meniscus modelling assumptions in a static tibiofemoral finite element model: importance of geometry over material.

Finite element studies of the tibiofemoral joint have increased use in research, with attention often placed on the material models. Few studies assess the effect of meniscus modelling assumptions in image-based models on contact mechanics outcomes. This work aimed to assess the effect of modelling assumptions of the meniscus on knee contact mechanics and meniscus kinematics. A sensitivity analysis was performed using three specimen-specific tibiofemoral models and one generic knee model. The assumptions in representing the meniscus attachment on the tibia (shape of the roots and position of the attachment), the material properties of the meniscus, the shape of the meniscus and the alignment of the joint were evaluated, creating 40 model instances. The values of material parameters for the meniscus and the position of the root attachment had a small influence on the total contact area but not on the meniscus displacement or the force balance between condyles. Using 3D shapes to represent the roots instead of springs had a large influence in meniscus displacement but not in knee contact area. Changes in meniscus shape and in knee alignment had a significantly larger influence on all outcomes of interest, with differences two to six times larger than those due to material properties. The sensitivity study demonstrated the importance of meniscus shape and knee alignment on meniscus kinematics and knee contact mechanics, both being more important than the material properties or the position of the roots. It also showed that differences between knees were large, suggesting that clinical interpretations of modelling studies using single geometries should be avoided.

Lithocholic Acid Alleviates Deoxynivalenol-Induced Inflammation and Oxidative Stress via PPARγ-Mediated Epigenetically Transcriptional Reprogramming in Porcine Intestinal Epithelial Cells.

Deoxynivalenol (DON) is a common mycotoxin that induces intestinal inflammation and oxidative damage in humans and animals. Given that lithocholic acid (LCA) has been suggested to inhibit intestinal inflammation, we aimed to investigate the protective effects of LCA on DON-exposed porcine intestinal epithelial IPI-2I cells and the underlying mechanisms. Indeed, LCA rescued DON-induced cell death in IPI-2I cells and reduced DON-stimulated inflammatory cytokine levels and oxidative stress. Importantly, the nuclear receptor PPARγ was identified as a key transcriptional factor involved in the DON-induced inflammation and oxidative stress processes in IPI-2I cells. The PPARγ function was found compromised, likely due to the hyperphosphorylation of the p38 and ERK signaling pathways. In contrast, the DON-induced inflammatory responses and oxidative stress were restrained by LCA via PPARγ-mediated reprogramming of the core inflammatory and antioxidant genes. Notably, the PPARγ-modulated transcriptional regulations could be attributed to the altered recruitments of coactivator SRC-1/3 and corepressor NCOR1/2, along with the modified histone marks H3K27ac and H3K18la. This study emphasizes the protective actions of LCA on DON-induced inflammatory damage and oxidative stress in intestinal epithelial cells via PPARγ-mediated epigenetically transcriptional reprogramming, including histone acetylation and lactylation.

Fast modulation of surface plasmons based on the photothermal effect of nonvolatile solid thin films.

Nonvolatile phase change materials owing to their robust stability and reversibility have shown significant potential in nanophotonic switches and memory devices. However, their performances deteriorate as the thickness decreases below 10 nm due to the local deformation induced by the phase change, which makes them less compatible with plasmonic nanogaps. Here, we address this issue by photothermally modulating the refractive index of germanium antimony telluride (GST) placed in plasmonic nanogaps, which tunes plasmon resonances in the visible region below the melting point of GST, making such optical switching highly reversible at a rate of up to hundreds of ∼kHz. They are also demonstrated to modulate the waveguiding efficiency of propagating surface plasmons, which is based on the photothermal modulation of plasmons with the assistance of GST. Such hybrid nanoplasmonic system with cost-effective fabrication and efficient operation method provides a promising route towards integrated nanophotonic chips.

Superplastic Nanomolding of Aluminum Waveguides for Subwavelength Light Routing, Splitting, and Encryption.

Plasmonic nanowires (NWs) due to their polarization-dependent optics and enhanced light-matter interactions have presented vibrant capabilities in functional nanophotonic devices. However, current demonstrations have largely been based on chemically synthesized Ag NWs, which are extremely unstable and poorly functional. Here we show single-crystalline Al NWs can be fabricated by a superplastic nanomolding (SPNM) technique on a centimeter scale, which are earth-abundant and highly stable. They present robust properties of multimode waveguiding with long-term stability, high efficiency of beam splitting in response to the polarization, and durable thermal optical modulation, which can be readily applied as nanophotonic routers, splitters, and information encryptors. Moreover, this SNPM technique is extendable to other metals, which are highly exploitable for functional nanophotonic devices and integrated optical chips.

Lactobacillus johnsonii N5 from heat stress-resistant pigs improves gut mucosal immunity and barrier in dextran sodium sulfate-induced colitis.

Developing effective strategies to prevent diarrhea and associated-gut disorders in mammals has gained great significance. Owing to the many health benefits provided by the commensal microbiota of the intestinal tract, such as against environmental perturbation, we explored the host phenotype-associated microbes and their probiotic potential. Based on the observations that the chronic heat stress-exposed weaned piglets present as heat stress-susceptible (HS-SUS) or heat stress-resistant (HS-RES) individuals, we confirmed the phenotypic difference between the two on growth performance (P < 0.05), diarrhea index (P < 0.001), intestinal heat shock protein 70 (HSP70) regulation (P < 0.01), and inflammatory responses (P < 0.01). By comparing the gut microbiome using 16S rRNA gene sequencing and KEGG functional analysis, we found that Lactobacillus johnsonii exhibited significantly higher relative abundance in the HS-RES piglets than in the HS-SUS ones (P < 0.05). Further experiments using a mouse model for chemical-induced inflammation and intestinal injury demonstrated that oral administration of a representative L. johnsonii N5 (isolated from the HS-RES piglets) ameliorated the clinical and histological signs of colitis while suppressing intestinal pro-inflammatory cytokines TNF-α and IL-6 production (P < 0.05). We found that N5 treatment enhanced tight junction proteins ZO-1 and occludin and cytoprotective HSP70 levels under physiological condition and restored their mucosal expressions in colitis (P < 0.05). In support of the high production of the anti-inflammatory cytokine IL-10, N5 promoted the intestinal Peyer's patches MHCII+ and CD103+ dendritic cell populations (P < 0.05), increased the regulatory T (Treg) cell numbers (P < 0.05), and decreased the Th17 population and its IL-17a production under physiological condition and during colitis (P < 0.01). Our results shed light on understanding the interaction between commensal Lactobacillus and the host health, and provide L. johnsonii N5 as an alternative to antibiotics for preventing diarrhea and intestinal diseases.

Plasmon-assisted nanophase engineering of titanium dioxide for improved performances in single-particle based sensing and photocatalysis.

Titanium dioxide (TiO2) due to its large bandgap, has a very limited efficiency in utilizing sunlight for photocatalysis and photoanode applications. Sensitizing with metallic nanoparticles is one of the promising routes for resolving this issue but it requires thermal annealing and proper bandgap engineering to optimize the Schottky junctions. Here we use plasmonic nanoheating to locally anneal the TiO2 medium with a sub-nanometer (sub-nm) feature, which results in a nanophase transition from amorphous TiO2 to anatase and rutile with a gradient configuration. Such gradient nanocoatings of rutile/anatase establish a cascade hot electron transfer via a conduction band and defect states, which improves the surface enhanced Raman scattering (SERS) performance and photocatalytic efficiency over an order of magnitude. Unlike conventional global annealing, this nanoannealing strategy with plasmonic heating enables sub-nm control at the interface between the metal and semiconductors, and this strategy not only provides new opportunities for single particle SERS, but also shows significant implications for photocatalysis and hot-electron chemistry.