A protective and broadly binding antibody class engages the influenza virus hemagglutinin head at its stem interface.

Influenza infection and vaccination impart strain-specific immunity that protects against neither seasonal antigenic variants nor the next pandemic. However, antibodies directed to conserved sites can confer broad protection. Here we identify and characterize a class of human antibodies that engage a previously undescribed, conserved epitope on the influenza hemagglutinin (HA) protein. Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal influenza infection in mice. Antibody isotypes that direct clearance of infected cells enhance this protection. Head-stem interface antibodies bind to most influenza A serotypes and seasonal human variants, and are present at low frequencies in the memory B cell populations of multiple human donors. Vaccines designed to elicit these antibodies might contribute to "universal" influenza immunity.

Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

Identification and functional analysis of LpNAC37 associated with somatic embryogenesis in Lilium pumilum DC. Fisch. based on transcriptome analysis.

Somatic embryogenesis (SE) is important for Lilium bulb propagation, germplasm conservation, and genetic transformation. The transition of somatic cells to embryonic cells is a critical step in SE, but the associated regulatory mechanisms have not been fully elucidated. Lilium pumilum DC. Fisch has a high regenerative capacity, and this study clarifies the critical timing of embryonic cell appearance in Lilium SE. Transcriptome sequencing using RNA-seq technology was performed on 5 representative samples from the early stage of Lilium SE. The 15 established cDNA libraries yielded 91.47 GB of valid data, and a total of 11,155 genes were consistently differentially expressed in the early stages of Lilium SE. GO annotation and KEGG pathway analysis of differentially expressed genes (DEGs) suggested that transcriptional regulation, hormone signaling, and stress response pathways play essential roles in the early stages of Lilium SE. WOX8, WOX11, SHR2, NAC37, AHP2, ANT, PIN1C, LAX2, LBD4, ACS12, YUC4, NFYB3, WRKY28, SAUR50, PYL9, and WRKY39 may be candidate genes for regulating early SE in Lilium. We further cloned LpNAC37, one of the key DEGs obtained from WGCNA and screening. LpNAC37 encodes a protein of 303 amino acids with a conserved NAM structural domain. The protein is a nuclear transcription factor with the highest homology to carrot DcNAC37. Overexpression of LpNAC37 suggested that LpNAC37 promotes embryonic callus formation in Arabidopsis. These results will help reveal the molecular mechanisms of the early stages of Lilium SE and advance the application of SE in Lilium propagation and genetic transformation.

Translation of therapeutic strategies to modulate B cell reponses from non-human primate models to human kidney transplantation.

Using novel drugs targeting lymphocyte costimulation, cytokines, antibody, complement, and plasma cells, we have developed strategies in a non-human primate model to modulate the B cell response to incompatible kidney transplants. After more than two decades of research supported by mechanistic studies, this has resulted in clinically relevant approaches that are currently enrolling in clinical trials or preparing for such. In this manner, we aim to address the problems of HLA sensitization for very highly sensitized patients awaiting transplantation and the unmet need of effective treatment for antibody-mediated rejection.

Recall of B cell memory depends on relative locations of prime and boost immunization.

Immunization or microbial infection can establish long-term B cell memory not only systemically but also locally. Evidence has suggested that local B cell memory contributes to early local plasmacytic responses after secondary challenge. However, it is unclear whether locality of immunization plays any role in memory B cell participation in recall germinal centers (GCs), which is essential for updating their B cell antigen receptors (BCRs). Using single B cell culture and fate mapping, we have characterized BCR repertoires in recall GCs after boost immunizations at sites local or distal to the priming. Local boosts with homologous antigen recruit the progeny of primary GC B cells to recall GCs more efficiently than do distal boosts. Recall GCs elicited by local boosts contain significantly more B cells with elevated levels of immunoglobulin (Ig) mutation and higher avidity BCRs. This local preference is unaffected by blocking CD40:CD154 interaction to terminate active, GC responses. Local boosts with heterologous antigens elicit secondary GCs with B cell populations enriched for cross-reactivity to the prime and boost antigens; in contrast, cross-reactive GC B cells are rare after distal boosts. Our results suggest that local B cell memory is retained in the form of memory B cells, GC B cells, and GC phenotype B cells that are independent of organized GC structures and that these persistent "primed B cells" contribute to recall GC responses at local sites. Our findings indicate the importance of locality in humoral immunity and inform serial vaccination strategies for evolving viruses.

Functional analysis of the eTM-miR171-SCL6 module regulating somatic embryogenesis in Lilium pumilum DC. Fisch.

Somatic embryogenesis (SE) is of great significance in Lilium bulb production, germplasm preservation and genetic improvement. miRNAs are important regulators of plant growth and development at the transcriptional level. Previous research by our group has shown that lpu-miR171 and its target gene SCARECROW-LIKE 6 (SCL6) play an important regulatory role in lily SE, and we predicted and identified that endogenous target mimics (eTMs) can regulate lpu-miR171. However, the associated mechanism and internal regulatory network are not yet clear. In the present study, lpu-miR171 was used as an entry point to explore the regulatory network between its upstream eTMs and its downstream target gene LpSCL6, as well as to identify the mechanism of this regulatory network in Lilium SE. Tobacco transient transformation confirmed that miRNA171 significantly inhibited the expression of LpSCL6. On this basis, the Lilium stable genetic transformation system was used to demonstrate that silencing lpu-miR171a and lpu-miR171b and overexpressing LpSCL6-II and LpSCL6-I promoted starch accumulation in calli and the expression of key cell cycle genes, thus providing energy to meet preconditions for SE and accelerate the formation and development of Lilium somatic embryos. LpSCL6-II and LpSCL6-I are nuclear proteins with self-activation activity in yeast cells. In addition, we confirmed in Lilium that lpu-eTM171 is the eTM of lpu-miR171 that binds lpu-miR171 to prevent cleavage of the target gene LpSCL6, thereby promoting SE. Therefore, the present study established a new mechanism whereby the eTM-miR171-SCL6 module regulates SE in Lilium pumilum DC. Fisch. and provided new insights clarifying the mechanism of SE.

A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.

Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.

Allo-Specific Humoral Responses: New Methods for Screening Donor-Specific Antibody and Characterization of HLA-Specific Memory B Cells.

Antibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.

Improvement of a Genetic Transformation System and Preliminary Study on the Function of LpABCB21 and LpPILS7 Based on Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Auxin transport mediates the asymmetric distribution of auxin that determines the fate of cell development. Agrobacterium-mediated genetic transformation is an important technical means to study gene function. Our previous study showed that the expression levels of LpABCB21 and LpPILS7 are significantly up-regulated in the somatic embryogenesis (SE) of Lilium pumilum DC. Fisch. (L. pumilum), but the functions of both genes remain unclear. Here, the genetic transformation technology previously developed by our team based on the L.pumilum system was improved, and the genetic transformation efficiency increased by 5.7-13.0%. Use of overexpression and CRISPR/Cas9 technology produced three overexpression and seven mutant lines of LpABCB21, and seven overexpression and six mutant lines of LpPILS7. Analysis of the differences in somatic embryo induction of transgenic lines confirmed that LpABCB21 regulates the early formation of the somatic embryo; however, excessive expression level of LpABCB21 inhibits somatic embryo induction efficiency. LpPILS7 mainly regulates somatic embryo induction efficiency. This study provides a more efficient method of genetic transformation of L. pumilum. LpABCB21 and LpPILS7 are confirmed to have important regulatory roles in L. pumilum SE thus laying the foundation for subsequent studies of the molecular mechanism of Lilium SE.

Full-Length Transcriptome Analysis of the ABCB, PIN/PIN-LIKES, and AUX/LAX Families Involved in Somatic Embryogenesis of Lilium pumilum DC. Fisch.

Plant cell totipotency is one of the 25 major topics in current scientific research, and somatic embryos are good experimental material for studying cell totipotency. Polar auxin transport plays an important regulatory role in somatic embryogenesis (SE). However, little is known about the auxin transport genes and their regulatory mechanisms in Lilium SE. In this study, we applied single-molecule real-time (SMRT) sequencing to Lilium pumilum DC. Fisch. for the first time and obtained a total of 119,649 transcripts, of which 14 encoded auxin transport genes. Correlation analyses between somatic embryo induction and gene expression under different treatments revealed that auxin transport genes, especially ATP-binding cassette (ABC) transporter B family member 21 (ABCB21) and PIN-FORMED (PIN) LIKES 7 (PILS7), may be key players in SE, and the necessary duration of picloram (PIC) treatment to induce SE is as short as 3 days. Our research provides valuable genetic information on Lilium pumilum, elucidating the candidate auxin transport genes involved in SE and their influencing factors. This study lays a foundation for elucidating the regulatory mechanism of auxin transport in SE.

Enhanced mechanical properties of 4H-SiC by epitaxial carbon films obtained from bilayer graphene.

Graphene exhibits excellent mechanical properties under atomically thin thickness, which made it very suitable for nanoelectromechanical systems that had high requirements for the thickness of coatings. The epitaxial bilayer graphene on the 4H-SiC (0001) surface presents high stiffness and hardness comparable to diamond. However, due to structural transition occurring at the nanoscale, it is difficult to elucidate reinforcement mechanisms using experimental methods. Here, we applied molecular dynamics simulations to study nanoindentation of epitaxial carbon-film-covered 4H-SiC (0001) surfaces. Because a weak interaction potential existed between graphene layers at indentation depth (h < 0.8 Å) that far smaller than interlayer distance, the epitaxial bilayer graphene does not allow the 4H-SiC to exceed its intrinsic stiffness. When the indentation depth h ≥6.45 Å, the sp3 hybridized bonds formed on the interlayer of graphene, which leads to fewer amorphous atoms in the sample of 4H-SiC and exhibits stronger stiffness, in comparison with bare 4H-SiC. This strongly suggests the existence of sp3 bonds contributing to the surface strengthening. Meanwhile, we found that the comprehensive mechanical properties of nanocomposites with hydrogenated diamond-like films were superior to those of nanocomposites with other carbon films at high temperatures.

Cross-Reactivity to Kynureninase Tolerizes B Cells That Express the HIV-1 Broadly Neutralizing Antibody 2F5.

2F5 is an HIV-1 broadly neutralizing Ab that also binds the autoantigens kynureninase (KYNU) and anionic lipids. Generation of 2F5-like Abs is proscribed by immune tolerance, but it is unclear which autospecificity is responsible. We sampled the BCR repertoire of 2F5 knock-in mice before and after the first and second tolerance checkpoints. Nearly all small pre-B (precheckpoint) and 35-70% of anergic peripheral B cells (postcheckpoint) expressed the 2F5 BCR and maintained KYNU, lipid, and HIV-1 gp41 reactivity. In contrast, all postcheckpoint mature follicular (MF) B cells had undergone L chain editing that purged KYNU and gp41 binding but left lipid reactivity largely intact. We conclude that specificity for KYNU is the primary driver of tolerization of 2F5-expressing B cells. The MF and anergic B cell populations favored distinct collections of editor L chains; surprisingly, however, MF and anergic B cells also frequently expressed identical BCRs. These results imply that BCR autoreactivity is the primary determinant of whether a developing B cell enters the MF or anergic compartments, with a secondary role for stochastic factors that slightly mix the two pools. Our study provides mechanistic insights into how immunological tolerance impairs humoral responses to HIV-1 and supports activation of anergic B cells as a potential method for HIV-1 vaccination.

Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism.

Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.

Small RNA and Transcriptome Sequencing Reveal a Potential miRNA-Mediated Interaction Network That Functions during Somatic Embryogenesis in Lilium pumilum DC. Fisch.

Plant somatic embryos are widely used in the fields of germplasm conservation, breeding for genetic engineering and artificial seed production. MicroRNAs (miRNAs) play pivotal roles in somatic embryogenesis (SE) regulation. However, their regulatory roles during various stages of SE remain unclear. In this study, six types of embryogenic samples of Lilium pumilum DC. Fisch., including organogenic callus, embryogenic callus induced for 4 weeks, embryogenic callus induced for 6 weeks, globular embryos, torpedo embryos and cotyledon embryos, were prepared for small RNA sequencing. The results revealed a total of 2,378,760 small RNA reads, among which the most common size was 24 nt. Four hundred and fifty-two known miRNAs, belonging to more than 86 families, 57 novel miRNAs and 40 miRNA*s were identified. The 86 known miRNA families were sorted according to an alignment with their homologs across 24 land plants into the following four categories: 23 highly conserved, 4 moderately conserved, 15 less conserved and 44 species-specific miRNAs. Differentially expressed known miRNAs were identified during various stages of SE. Subsequently, the expression levels of 12 differentially expressed miRNAs and 4 targets were validated using qRT-PCR. In addition, six samples were mixed in equal amounts for transcript sequencing, and the sequencing data were used as transcripts for miRNA target prediction. A total of 66,422 unigenes with an average length of 800 bp were assembled from 56,258,974 raw reads. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that 38,004 and 15,497 unigenes were successfully assigned to GO terms and KEGG pathways, respectively. Among the unigenes, 2,182 transcripts were predicted to be targets for 396 known miRNAs. The potential targets of the identified miRNAs were mostly classified into the following GO terms: cell, binding and metabolic process. Enriched KEGG analysis demonstrated that carbohydrate metabolism was the predominant pathway in Lilium SE. Thus, we performed systemic characterization, homology comparisons and profiling of miRNA expression, and we constructed an miRNA-target network during Lilium SE for the first time. Our findings establish a foundation for the further exploration of critical genes and elucidation of SE in Lilium.

Automatic segmentation of myocardium from black-blood MR images using entropy and local neighborhood information.

By using entropy and local neighborhood information, we present in this study a robust adaptive Gaussian regularizing Chan-Vese (CV) model to segment the myocardium from magnetic resonance images with intensity inhomogeneity. By utilizing the circular Hough transformation (CHT) our model is able to detect epicardial and endocardial contours of the left ventricle (LV) as circles automatically, and the circles are used as the initialization. In the cost functional of our model, the interior and exterior energies are weighted by the entropy to improve the robustness of the evolving curve. Local neighborhood information is used to evolve the level set function to reduce the impact of the heterogeneity inside the regions and to improve the segmentation accuracy. An adaptive window is utilized to reduce the sensitivity to initialization. The Gaussian kernel is used to regularize the level set function, which can not only ensure the smoothness and stability of the level set function, but also eliminate the traditional Euclidean length term and re-initialization. Extensive validation of the proposed method on patient data demonstrates its superior performance over other state-of-the-art methods.

Full screening and accurate subtyping of HLA-A*02 alleles through group-specific amplification and mono-allelic sequencing.

HLA-A*02 is the most prevalent and polymorphic major histocompatibility complex (MHC) allele family in humans. Functional differences have been revealed among subtypes, demanding further subtyping of HLA-A*02 in basic and clinical settings. However, the fast growing polymorphisms render traditional primer- or probe-based typing methods impractical and result in increasing ambiguities in direct sequence-based typing. In this study, we combined group-specific amplification and mono-allelic sequencing to design and validate a simple scheme for the complete screening and accurate subtyping of all 540 reported HLA-A*02 alleles. This scheme could be performed in routine labs to facilitate studies with an interest in HLA-A*02.

Human leukocyte antigen E in human cytomegalovirus infection: friend or foe?

Human cytomegalovirus (HCMV) is a well-studied ß-herpesvirus virus, which adopts a variety of strategies to evade immune surveillance. It has been reported that in HCMV-infected cells, classical major histocompatibility (MHC) class I molecules are down-regulated, but the MHC class Ib molecule human leukocyte antigen (HLA)-E is normally expressed or even overexpressed on the cell surface. HLA-E has been first described to interact with CD94/NKG2 receptors expressed mainly on the surface of natural killer (NK) cells, thus confining its role to the regulation of NK-cell function. The engagement of CD94/NKG2A with HLA-E, with a signal peptide of the HCMV glycoprotein UL40, usually induces inhibitory signals. However, HLA-E also serves as a ligand for the TCR expressed by αßCD8(+) T cells. Recognition of peptides presented by HLA-E may result in CD8(+) effector T-cell activation. These findings will help to understand more on both pathogenic and protective roles of HLA-E in HCMV infection. In this review, we discussed recent studies about the roles of HLA-E in HCMV infection.

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of I(to) and augmentation of K(ATP) channels.

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.

Effect of polydatin on action potential in ventricular papillary muscle of rat and the underlying ionic mechanism.

It is proved that polydatin has cardioprotection against ischemia-induced arrhythmia, but the electrophysiological mechanism is not clear. The aim of the present study was to investigate the effect of polydatin on action potential (AP) in ventricular papillary muscle and the underlying ionic mechanism in rat using intracellular recording and whole-cell patch clamp techniques. The results showed: (1) In normal papillary muscles, polydatin (50 and 100 µmol/L) shortened duration of 50% repolarization (APD(50)) and duration of 90% repolarization (APD(90)) in a concentration-dependent manner (P<0.01). But polydatin had no effects on resting potential (RP), overshoot (OS), amplitude of action potential (APA) and maximal rate of depolarization in phase 0 (V(max)) in normal papillary muscles (P>0.05). (2) In partially depolarized papillary muscles, polydatin (50 µmol/L) not only shortened APD(50) and APD(90) (P<0.05), but also decreased OS, APA and V(max) (P<0.05). (3) After pretreatment with glibenclamide (10 µmol/L), an ATP-sensitive K(+) channel blocker, the electrophysiological effect of polydatin (50 µmol/L) was partially inhibited. (4) Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L), a nitric oxide (NO) synthase inhibitor, failed to abolish the effect of polydatin (50 µmol/L) on AP. (5) Polydatin (25, 50, 75 and 100 µmol/L) decreased L-type Ca(2+) current in ventricular myocytes in a concentration-dependent manner (P<0.05). (6) Polydatin (50 µmol/L) increased ATP-sensitive K(+) current in ventricular myocytes (P<0.05). The results suggest that polydatin can shorten the repolarization of AP in normal papillary muscle and inhibit AP in partially depolarized papillary muscle, which might be related to the blocking of L-type Ca(2+) channel and the opening of ATP-sensitive K(+) channel.

Thymic regulation of autoimmune disease by accelerated differentiation of Foxp3+ regulatory T cells through IL-7 signaling pathway.

The exact role of adult thymus in autoimmune disease state is poorly understood. We show here that thymus regulated experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, as evidenced by loss of spontaneous recovery in thymectomized EAE mice. There was progressive enrichment for CD4 single-positive Foxp3(+) regulatory T cells in thymocytes during the course of EAE and they suppressed the disease when adoptively transferred. Thymus was shown to undergo an active process characterized by accelerated differentiation and proliferation of regulatory T (Treg) cells through a mechanism involving increased expression of IL-7 in stromal cells and dynamic expression of IL-7 receptor in thymic Treg cells. This process preceded EAE recovery and selectively affected Treg over non-Treg cells in the thymus, leading to increased output of thymic Treg cells and self-regulation of EAE. The study reveals a novel role of thymus in self-regulation of autoimmune condition.