Search

Supplementary Methods and Figure Legends from Mycoplasma Hyorhinis Infection Promotes NF-κB–Dependent Migration of Gastric Cancer Cells

Graphene oxide (GO) has been widely used as a multi-functional filler in various composites. In order to improve the properties of room temperature vulcanized (RTV) silicone rubber, the functionalized GO (FGO) was used as 2-dimensional filler with short carbon fiber (SCF) and silica particle. The effects of the FGO content on the properties of the RTV silicone rubber composites were investigated. When appropriate amount of the FGOs is added in the composite, the interfacial bonding between SCFs and silicone rubber matrix could be effectively enhanced and the properties of the composites are improved. For the composite with 0.5 phr FGO, the tensile and tear strengths are about 4.7 MPa and 21.9 kN/m, respectively. Meanwhile, the corresponding values of the composite without FGO are only 4.0 MPa and 20.3 kN/m, respectively. The above said values of the composite with 1 phr FGO are 3.8 MPa and 19.8 kN/m, respectively. In addition, the onset decomposition temperature and temperature at the highest decomposition rate for the composite with 0.5 phr FGO, both remarkably increase from 493.4 to 504.2 °C and 564.4 to 613.1 °C, respectively. The carbon frameworks (dense layer and porous layer) will form during the ablation process and play the key role in protecting the RTV silicone rubber, which leads to the reduction of the ablative rate.

Rehmannia glutinosa has important medicinal value; terpenoid is one of the main active components in R. glutinosa. In this study, iTRAQ technique was used to analyze the relative abundance of proteins in roots of R. glutinosa, and 6,752 reliable proteins were quantified. GO enrichment results indicated that most proteins were involved in metabolic process or cellular process, 57.63% proteins had catalytic activity, and 65.80% proteins were enriched in membrane-bounded organelle. In roots of R. glutinosa, there were 38 KEGG enrichments with significance, more DEPs were found in some pathways, especially the proteasome pathway and TCA cycle with 15.0% DEPs between elongation stage and expansion stage of roots. Furthermore, five KEGG pathways of terpenoid synthesis were found. Most prenyltransferases belong to FPP/GGPP synthase family, involved in terpenoid backbone biosynthesis, and all interacted with biotin carboxylase CAC2. Compared with that at the elongation stage, many prenyltransferases exhibited higher expression at the expansion stage or maturation stage of roots. In addition, eight FPP/GGPP synthase encoding genes were cloned from R. glutinosa, namely FPPS, FPPS1, GGPS, GGPS3, GGPS4, GGPS5, GPPS and GPPS2, introns were also found in FPPS, FPPS1, GGPS5 and GGPS2, and FPP/GPP synthases were more conservative in organisms, especially in viridiplantae, in which the co-occurrence of GPPS or GPPS2 was significantly higher in plants. Further analysis found that FPP/GGPP synthases of R. glutinosa were divided into three kinds, GGPS, GPPS and FPPS, and their gene expression was significantly diverse in different varieties, growth periods, or tissues of R. glutinosa. Compared with that of GGPS, the expression of GPPS and FPPS was much higher in R. glutinosa, especially at the expansion stage and maturation stage. Thus, the synthesis of terpenoids in roots of R. glutinosa is intricately regulated and needs to be further studied.

As the traditional Chinese herb, 'Rehmannia glutinosa' ('R.glutinosa') has significant effects on health. The main active ingredient of 'R. glutinosa' is iridoid. In this study, root, stem and leaf of 'R. glutinosa' at five different growth stages were collected, the content of iridoid in 'R. glutinosa' was determinated, and the expression of key enzymes in 'R. glutinosa' was analyzed by quantitative real-time PCR (q-PCR). We found that the content of iridoid was increased continuously during the first three growth stages (I-III) of 'R. glutinosa'. It reached the maximum value in root and leaf at growth stage III of 'R. glutinosa'. However, the content of iridoid was not lower in leaf, compared with root. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), Geranyl pyrophosphate synthase (GPPS), Geraniol 10-hydroxylase (G10H) and 10-hydroxygeraniol oxidoreductase (10HGO) were the key enzymes involved in the synthesis of iridoid in plant, but their expression pattern or level was different in various tissues and stages of 'R. glutinosa'. All appeared tissue specificity and their expression was related to the accumulation of iridoid in 'R. glutinosa'. Thus, DXR, GPPS, G10H and 10HGO might take part in the synthesis of iridoid in 'R. glutinosa' and could be differently regulated during the growth and development of 'R. glutinosa', which would provide theoretical basis for the research on secondary metabolism of iridoid in R. glutinosa.

In August 2014, a pit containing more than 10000 porcelain shards was excavated in the southwestern area of the Palace Museum. Reign marks and other stylistic criteria dated the manufacture of thes...

Background: Epigenetic regulation plays a significant role in the accumulation of plant secondary metabolites. The terpenoids are the most abundant in the secondary metabolites of plants, iridoid glycosides belong to monoterpenoids which is one of the main medicinal components of R.glutinosa. At present, study on iridoid glycosides mainly focuses on its pharmacology, accumulation and distribution, while the mechanism of its biosynthesis and the relationship between DNA methylation and plant terpene biosynthesis are seldom reports. Results: The research showed that the expression of DXS, DXR, 10HGO, G10H, GPPS and accumulation of iridoid glycosides increased at first and then decreased with the maturity of R.glutinosa, and under different concentrations of 5-azaC, the expression of DXS, DXR, 10HGO, G10H, GPPS and the accumulation of total iridoid glycosides were promoted, the promotion effect of low concentration (15μM-50μM) was more significant, the content of genomic DNA 5mC decreased significantly, the DNA methylation status of R.glutinosa genomes was also changed. DNA demethylation promoted gene expression and increased the accumulation of iridoid glycosides, but excessive demethylation inhibited gene expression and decreased the accumulation of iridoid glycosides. Conclusion: The analysis of DNA methylation, gene expression, and accumulation of iridoid glycoside provides insights into accumulation of terpenoids in R.glutinosa and lays a foundation for future studies on the effects of epigenetics on the synthesis of secondary metabolites.

SUMMARY Soluble “SOSIP”-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high-titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the effect on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, here, we quantify the prevalence of trimer-base antibody responses in 49 non-human primates immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. Trimer-base responses account for ~90% of the overall trimer response in animals immunized with trimer only, ~70% in animals immunized with a cocktail of SOSIP trimer and FP conjugate, and ~30% in animals primed with FP conjugates before trimer immunization. Notably, neutralization breadth in FP-conjugate-primed animals correlates inversely with trimer-base responses. Our data provide methods to quantify the prevalence of trimer-base responses and reveal that FP-conjugate priming, either alone or as part of a cocktail, can reduce the trimer-base response and improve the neutralization outcome., In brief The exposed base region of soluble HIV-1 Env trimers elicits strong non-neutralizing antibody responses. Corrigan et al. quantify plasma anti-base responses in immunized NHPs and observe a reduction in anti-base responses with fusion-peptide priming. The percentage of anti-base responses correlates inversely with neutralization breadth, providing insights for improving vaccination strategies., Graphical Abstract

Background: The growth and development of wheat are seriously influenced by drought stress, and the research on drought resistance mechanism of wheat is very important. Dehydration responsive element binding protein (DREB) plays an important role in plant response to drought stress, but epigenetic regulation for gene expression of DREB transcription factor is less studied, especially the regulatory role of DNA methylation has not been reported.Results: In this research, DREB2, DREB6 and Wdreb2 were cloned from wheat in this study, their CDS sequence was composed of 732bp, 837bp or 1035bp, respectively, one 712bp intron was found in DREB6. Although AP2/EREBP domain of DREB2, DREB6 and Wdreb2 had 73.25% identity, they belong to different types of DREB transcription factor, and the expression of Wdreb2 was significantly higher, yet was the lowest in DREB2. Under drought stress, the expression of DREB2, DREB6 and Wdreb2 could be induced, but had different trends along with the increase of stress time, and their expression had tissue specificity, was obviously higher in leaf. Promoter of DREB2, DREB6 and Wdreb2 in leaf was further studied, some elements related to adverse stress were found, and the promoter of DREB2 and Wdreb2 was slightly methylated, but DREB6 promoter was mildly methylated. Compared with the control, the level of promoter methylation decreased in DREB2 and DREB6 as stressed for 2h, then increased along with the increase of stress time, which was opposite in Wdreb2 promoter, the status of promoter methylation also had significant change under drought stress. Further analysis showed that promoter methylation of DREB6 or Wdreb2 was negatively correlated with their expression, especially was significant in Wdreb2. Conclusions: DREB2, DREB6 and Wdreb2 might function differently in response to drought stress, and promoter methylation had more significant effects on gene expression of Wdreb2 and DREB6.

In this study, ITS, ITS2, matK, rbcL and psbA-trnH in Rehmannia were successfully amplified and sequenced, but some ITS sequences need to be proofread according to ITS2 sequences. Compared with rbcL, matK and psbA-trnH, ITS and ITS2 had higher mutation rate and more information sites, and ITS2 had higher interspecific diversity and lower intraspecific variation in Rehmannia, but the interspecific genetic variation of rbcL and matK was lower. Furthermore, the obvious barcoding gap was found in psbA-trnH or ITS2 + psbA-trnH, and the overlap between interspecific and intraspecific variation of ITS, ITS2 or matK was less. In addition, the phylogenetic tree based on ITS or ITS2 indicated that R. glutinosa, R. chingii or R. henryi with obvious monophyly could be successfully identified, but R. piasezkii and R. elata were clustered into one branch, R. solanifolia could not be distinguished from R. glutinosa, and R. chingii was closer to R. henryi. In phylogenetic tree based on psbA-trnH or ITS2 + psbA-trnH, cultivars and wild varieties of R. glutinosa could be distinguished, were clearly separated from other Rehmannia species, and cultivars or wild varieties of R. glutinosa could be also distinguished by matK. Taken together, ITS2 has great potential in systematic study and species identification of Rehmannia, the combination of ITS2 and psbA-trnH might be the most suitable DNA barcode for Rehmannia species.

Objective Epithelial ovarian cancer (EOC) is the deadliest type of ovarian cancer, but the mechanisms contributing to its tumorigenesis are not well understood. Herein, we will elucidate the role of Ten-eleven translocation 1 (TET1) in EOC development. Methods The expression of TET1 in EOC cell lines and primary samples was examined by western blot and immunohistochemistry. The biological role of ectopic TET1 overexpression was revealed by a series of in vitro functional studies. Its downstream signaling pathway was predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of microarray data. The methylation level and expression of Wnt/β-catenin signaling inhibitors Dikkopf 1 (DKK1) and secreted Fzd receptor protein 2 (SFRP2) were examined by Chromatin immunoprecipitation (ChIP) assay, Epimark™ 5hmC and 5mC level analysis and quantitative RT-PCR. Small interference RNA (siRNA) technology was used to investigate the biological roles of DKK1 and SFRP2. Results TET1 expression was inversely correlated with clinical stage in patients with EOC by tissue microarray (TMA). TET1 expression was undetected in 6 types of EOC cell lines. Ectopic expression of TET1 inhibited colony formation, cell migration and invasion in SKOV3 and OVCAR3 cells. Furthermore, TET1 overexpression reversed the epithelial-mesenchymal transition (EMT) process of SKOV3 cells. Mechanistically, TET1 potently inhibited canonical Wnt/β-catenin signaling by demethylating and upregulating two upstream antagonists of this pathway, SFRP2 and DKK1, which was associated with inhibition of EMT and cancer cell metastasis. Conclusion This study uncovers that TET1 has potent tumor-suppressive effects in EOC by activating Wnt/β-catenin signaling inhibitors DKK1 and SFRP2.

Dehydration-responsive element-binding protein (DREB) plays an important role in response to osmotic stress. In this study, DREB2, DREB6 and Wdreb2 are isolated from wheat AK58, yet they belong to different types of DREB transcription factors. Under osmotic stress, the transcript expression of DREB2, DREB6 and Wdreb2 has tissue specificity and is generally higher in leaves, but their expression trends are different along with the increase of osmotic stress. Furthermore, some elements related to stresses are found in their promoters, promoters of DREB2 and Wdreb2 are slightly methylated, but DREB6’s promoter is moderately methylated. Compared with the control, the level of promoter methylation in Wdreb2 is significantly lower under osmotic stress and is also lower at CG site in DREB2, yet is significantly higher at CHG and CHH sites in DREB2, which is also found at a CHG site in DREB6. The status of promoter methylation in DREB2, DREB6 and Wdreb2 also undergoes significant changes under osmotic stress, further analysis showed that promoter methylation of Wdreb2 is negatively correlated with their expression. Therefore, the results of this research suggest the different functions of DREB2, DREB6 and Wdreb2 in response to osmotic stress and demonstrate the effects of promoter methylation on the expression regulation of Wdreb2.

The focus of this paper is on analysis, comparison and research on the colorful low-temperature, lead-containing overglazes on glazed porcelain body and on the enamel glazes on the metal body of the Qing Dynasty by adopting several analytical methods. Analysis and tests on the element, boron in overglaze on glazed porcelain body and enamel glaze on metal body, were performed using laser induced breakdown spectroscopy (LIBS), and the results showed that Cloisonne enamel, painted enamel and Falangcai samples contained boron, while Famille Rose (Fencai) samples did not contain boron. Meanwhile, such analysis methods as laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), energy dispersive X-ray spectroscopy (EDXRF), Micro-Raman, stereomicroscope and Confocal laser scanning microscopy (CLSM) were used to test and observe the element composition, crystal composition and microstructure of the samples. The results illustrated that matrix glaze of Cloisonne enamel, painted enamel and Falangcai was the same. The yellow glaze was a lead-alkali glass and other color glazes were boron-lead-alkali glass, while all color glazes of Famille Rose were lead-alkali glass. Colorful low-temperature overglaze on glazed porcelain body and enamel glaze on metal body had a common practice and technology in the use of opacifiers and colorants. Compared to painted enamel, the painting technique of Famille Rose was more complicated, and effect was apparently praised as being superior.

Vaccine elicitation of broadly neutralizing antibodies (bnAbs) is a key HIV-research goal. The VRC01 class of bnAbs targets the CD4-binding site on the HIV-envelope trimer and requires extensive somatic hypermutation (SHM) to neutralize effectively. Despite substantial progress, vaccine-induced VRC01-class antibodies starting from unmutated precursors have exhibited limited neutralization breadth, particularly against viruses bearing glycan on loop D residue N276 (glycan276), present on most circulating strains. Here, using sequential immunization of immunoglobulin (Ig)-humanized mice expressing diverse unmutated VRC01-class antibody precursors, we elicited serum responses capable of neutralizing viruses bearing glycan276 and isolated multiple lineages of VRC01-class bnAbs, including two with >50% breadth on a 208-strain panel. Crystal structures of representative bnAbs revealed the same mode of recognition as known VRC01-class bnAbs. Structure-function studies further pinpointed key mutations and correlated their induction with specific immunizations. VRC01-class bnAbs can thus be matured by sequential immunization from unmutated ancestors to >50% breadth, and we delineate immunogens and regimens inducing key SHM.

Longquan celadon is one of the most valuable treasures in Chinese ceramic history. Representative products are Ge ware (Ge meaning elder brother, black body celadon) and Di ware (Di meaning younger brother, gray body celadon) of the Song Dynasty (960–1279 A.D.). In this study, Ge and Di ware shards excavated from Wayaoyang kiln site in Longquan were collected and studied. Chemical and crystallite composition, microstructure, body and glaze thickness, firing temperature and glaze reflectance spectrum were observed and examined. Differences in raw materials and manufacturing technology between Ge and Di ware were studied. Based on the results and historical background, it was speculated that some Ge wares from Wayaoyang kiln site might be the test products of jade-like black body celadon for the imperial court.

Room temperature vulcanized (RTV) silicone rubber composites reinforced by multi-walled carbon nanotubes (MWCNTs) were prepared. The effect of surface modification methods and MWCNT contents on the properties of the RTV silicone rubber composites was investigated. The properties of the composites were improved when the treated MWCNTs were used. After treated by polyester-modified polydimethylsiloxane solution (BKY-310), the MWCNTs were uniformly dispersed in the silicone rubber matrix. When 5 phr of MWCNTs was added, the composite achieved good comprehensive performance. Its tensile strength, tear strength, elongation and onset decomposition temperature reach 2.0 MPa, 11.7 kN/m, 238% and 510 °C, respectively. But the above said values for the composite with untreated MWCNTs are only 1.1 MPa, 7.0 kN/m, 83% and 484 °C, respectively.

Summary: The HIV fusion peptide (FP) is a promising vaccine target. FP-directed monoclonal antibodies from vaccinated macaques have been identified that neutralize up to ∼60% of HIV strains; these vaccinations, however, have involved ∼1 year with an extended neutralization-eclipse phase without measurable serum neutralization. Here, in 32 macaques, we test seven vaccination regimens, each comprising multiple immunizations of FP-carrier conjugates and HIV envelope (Env) trimers. Comparisons of vaccine regimens reveal FP-carrier conjugates to imprint cross-clade neutralizing responses and a cocktail of FP conjugate and Env trimer to elicit the earliest broad responses. We identify a signature, appearing as early as week 6 and involving the frequency of B cells recognizing both FP and Env trimer, predictive of vaccine-elicited breadth ∼1 year later. Immune monitoring of B cells in response to vaccination can thus enable vaccine insights even in the absence of serum neutralization, here identifying FP imprinting, cocktail approach, and early signature as means to improve FP-directed vaccine responses.

The quality and yield of wheat (Triticum aestivum L.) are dramatically affected by drought. We used morphological and physiological characteristics and degree of DNA methylation to compare the responses of two wheat cultivars under osmotic stress, and found that the two cultivars behaved differently. Root development, leaf growth, and the accumulation of proline and soluble carbohydrate in wheat cv. AK58 all showed drought tolerance. Drought tolerance of wheat cv. XM13 was mainly improved by accumulation of proline and soluble carbohydrate. The degree of DNA methylation in wheat showed tissue specificity and increased significantly in leaf tissue with increasing osmotic stress, but decreased significantly in root tissue under mild osmotic stress. In addition, changes of DNA methylation differed between two wheat cultivars under osmotic stress, and this change was especially significant in AK58. Therefore, wheat AK58 may have stronger self-adjustment ability under osmotic stress compared with XM13, and might respond more rapidly to osmotic stress through the change of DNA methylation. This finding could be significant for revealing drought-tolerance mechanisms of plants.

Summary An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

Vaccine reverse engineering is emerging as an important approach to vaccine antigen identification, recently focusing mainly on structural characterization of interactions between neutralizing monoclonal antibodies (mAbs) and antigens. Using mAbs that bind unknown antigen structures, we sought to probe the intrinsic features of antibody antigen-binding sites with a high complexity peptide library, aiming to identify conformationally optimized mimotope antigens that capture mAb-specific epitopes. Using a high throughput sequencing-enhanced messenger ribonucleic acid (mRNA) display approach, we identified high affinity binding peptides for a hepatitis C virus neutralizing mAb. Immunization with the selected peptides induced neutralizing activity similar to that of the original mAb. Antibodies elicited by the most commonly selected peptides were predominantly against specific epitopes. Thus, using mRNA display to interrogate mAbs permits high resolution identification of functional peptide antigens that direct targeted immune responses, supporting its use in vaccine reverse engineering for pathogens against which potent neutralizing mAbs are available. Research in Context We used a large number of randomly produced small proteins (“peptides”) to identify peptides containing specific protein sequences that bind efficiently to an antibody that can prevent hepatitis C virus infection in cell culture. After the identified peptides were injected into mice, the mice produced their own antibodies with characteristics similar to the original antibody. This approach can provide previously unavailable information about antibody binding and could also be useful in developing new vaccines., Highlights • mRNA-display/high throughput sequencing identified high affinity peptide binders to monoclonal antibody (mAb). • The profile of selected peptides characterized the binding specificity of the selection mAb. • Immune responses induced by selected peptides were neutralizing and epitope-focused, mimicking the selection mAb.

Our previous works showed chronic infection of Mycoplasma hyorhinis (M. hyorhinis) was associated with gastric cancer metastasis, but the mechanisms were unknown. Herein, we found M. hyorhinis induced epithelial-mesenchymal transition (EMT) in gastric cancer cell MGC803, which was counteracted by inhibitor of NF-κB signaling or p65 knockdown. Furthermore, we found that TLR4 associated with p37, a membrane protein of M. hyorhinis. Knock-down or inhibition of TLR4 antagonized M. hyorhinis-induced NF-κB signaling, EMT, and cell migration. Thus, M. hyorhinis induces EMT and promotes cell migration via TLR4-NF-κB signaling, which provides a clue to the pathogenesis of M. hyorhinis in gastric cancer.

Disease diagnosis can be performed based on fusing the data acquired by multiple medical sensors from patients, and it is a crucial task in sensor-based e-healthcare systems. However, it is a challenging problem that there are few effective diagnosis methods based on sensor data fusion for atrial hypertrophy disease. In this article, we propose a novel multi-sensor data fusion method for atrial hypertrophy diagnosis, namely, characterized support vector hyperspheres (CSVH). Instead of constructing a hyperplane, as a traditional support vector machine does, the proposed method generates “hyperspheres” to collect the discriminative medical information, since a hypersphere is more powerful for data description than a hyperplane. In detail, CSVH constructs two characterized hyperspheres for the classes of patient and healthy subject, respectively. The hypersphere for the patient class is developed in a weighted version so as to take the diversity of patient instances into consideration. The hypersphere for the class of healthy people keeps furthest away from the patient class in order to achieve maximum separation from the patient class. A query is labelled by membership functions defined based on the two hyperspheres. If the query is rejected by the two classes, the angle information of the query to outliers and overlapping-region data is investigated to provide the final decision. The experimental results illustrate that the proposed method achieves the highest diagnosis accuracy among the state-of-the-art methods.

Two Cloisonné enamel architectural components from Fuwangge in the Forbidden City that were produced from Yangzhou (one production center) in Qing Dynasty (1616-1911 A.D.) were chosen and analyzed. A combination of Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and micro-Raman spectroscopy was successfully used to analyze eight colors in enamel glazes (yellow, white, pink, turquoise, yellow green, deep blue, red and deep green). Chemical composition results reveal that the enamel glaze matrix belongs to lead-potash-lime glass (PbO-K2O-CaO-SiO2). Based on Raman spectroscopy, lead-tin yellow types II, cassiterite, lead arsenate, fluorite and hematite were found as opacifiers and/or colorants. In addition, a detailed discussion of raw materials, such as fluorite and borax, might provide valuable information to trace manufacturing technology and provenance.

Worldwide >150 million individuals are chronically infected with hepatitis C virus (HCV) and ~3.2 million are infected in the USA alone (1). Studies show that 60–85% of acute infections become chronic and it has been determined that ~15–20% of all individuals that become infected with HCV progress to serious end-stage liver disease, such as cirrhosis or hepatocellular carcinoma (2). With ~18,000 new HCV infections occurring in the U.S. annually (3) the development of a vaccine against this virus is imperative. Although HCV persists in the majority of cases, acute infections are spontaneously resolved in ~20–25% of humans (4) and ~40% of chimpanzees (5). Chimpanzees represent the only animal model available to study pathogenesis of this viral disease and replicate the features of HCV infection in humans, such as kinetics of viremia and persistent infection even in the presence of immune responses (6). Spontaneous clearance following primary infection with HCV has been associated with effective T-cell responses, the major characteristics of which have been shown to be strong proliferating capacity of HCV-specific T-cells (7, 8), higher frequencies of CD8+ T-cells expressing perforin in the liver (9) and early and multispecific IFNγ-production by CD4+ and CD8+ T-cells (10, 11). Due to this association, several chimpanzee prophylactic vaccine studies have focused on T-cell induction targeting non-structural proteins for prevention of chronic HCV infection. Although these T-cell-based studies confirm that vaccine-induced T-cell responses can lead to memory cells and early control of viral replication (5) there is no greater incidence of viral clearance in vaccinees compared to naive animals when non-structural components are included in vaccines (5). In addition, we have previously shown that an ineffective T-cell vaccine can create greater pressure for viral mutation and therefore immune escape, which may lead to persistence of HCV (12). The failure of most T-cell vaccines to result in clearance of HCV is at odds with the knowledge that following spontaneous clearance and re-infection, viremia and liver disease are significantly reduced in both intensity and duration in chimpanzees (13, 14) and humans (4) and that this rapid clearance appears to be associated with faster T-cell activation (7, 14). We wished to assess if vaccine-induced T-cell responses differ qualitatively from those induced by natural infection and if memory T-cells with specific phenotypes in immune-primed animals can be associated with rapid and successful clearance of HCV. The initiation of the immune response starts with activation of T-cell receptors which leads to a signal cascade, up-regulation of activation markers and cellular proliferation. Analyses of hepatic gene expression in chimpanzees have suggested that efficient T-cell activation or recruitment is critical for HCV clearance during primary infections (15). The surface markers CD38 and/or HLA-DR have been used as measures of T-cell activation in many studies of acute and chronic infections, including HCV (16, 17), and during studies of yellow fever and smallpox vaccinees (18). These two markers in combination with Ki-67 and Bcl2 have been suggested as a means to identify CD8+ effector T-cell responses after immunization or infection (18). Ki-67 is a marker of proliferation in T-cells (19) while reduced Bcl2 expression indicates susceptibility to apoptosis; resting T-cells express high levels of the protein (20). We analyzed the frequency and kinetics of these markers of activation and proliferation on CD4+ and CD8+ T-cells in chimpanzees that were previously infected with HCV and had spontaneously cleared infection, vaccinated and control chimpanzees before and after infection with HCV and correlated this data with rapid control of the virus or progression to persistent infection.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro . However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W 437 , L 438 , L 441 , and F 442 , within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo .

Verbascoside is a phenylethanoid glycoside found in many plant species, and possessing some pharmacologically beneficial activities for human health. The tentative pathway and early steps of verbascoside biosynthesis have been documented, but key enzymes and their corresponding genes await discovery. We aimed to discover and analyze verbascoside biosynthesis-associated genes. The combined Rehmannia glutinosa tuberous root transcriptome was sequenced de novo. Some verbascoside biosynthesis-associated genes were discovered by the informatics method, validated by RT-PCR and analyzed by qRT-PCR. 149.8 million sequencing reads were generated and assembled de novo, yielding 96,961 final unigenes. Of them, 11,848 unigenes were mapped to 280 KEGG pathways, and 19 genes were assigned to the verbascoside biosynthesis pathway by comparing KEGG pathways and the tentative pathway. Eight genes were validated using RT-PCR in the dataset; qRT-PCR analysis showed that they were expressed in tuberous roots and leaves of both high and low verbascoside R. glutinosa cultivars, and their expressions were greater in the high verbascoside cultivar than in the low one, in old tuberous roots than in young ones, and in leaves than in tuberous roots. The differences in their relative expression levels suggest decisive roles for these enzymes in verbascoside biosynthesis. The results will accelerate the understanding of the genetic basis of verbascoside biosynthesis and genetic engineering breeding in R. glutinosa.

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2∗02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2∗02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

In this research, callus from leaves, petioles and stems of Achyranthes bidentata was evidently initiated by plant growth substance, in which 2,4-dichlorophenoxyacetic acid (2,4-D) was very important to callus induction, but effects of other plant growth substances were various, and the optimum combination of plant growth substances for callus induction from leaves, petioles and stems was respectively obtained. Compared with callus induction from leaves and petioles, callus induction from stems was easier, and the higher induction rate and bigger mass of callus from stems were obtained. This study showed that the dedifferentiation capacity of various explants from Achyranthes bidentata was obviously different, and effects of plant growth substance on callus induction from various explants of Achyranthes bidentata were significantly diverse.