PepMapper: a collaborative web tool for mapping epitopes from affinity-selected peptides

Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/     

A genotyping platform assembled with high-throughput DNA extraction,codominant functional markers,and automated CE system to accelerate marker-assisted improvement of rice

The introgression of multiple genes into traditional cultivars using marker-assisted selection (MAS) in order to obtain favorable traits is an effective strategy to achieve improved rice lines. Genotyping of markers is a central component of the evaluation of germplasm and the selection of progeny lines. However, efficient DNA extraction and genotyping of large breeding populations still remain limiting factors in rice molecular breeding programs. This study has developed and validated a cost-effective, rapid (<1 h for 96 samples), and high-throughput (96-well format) total DNA-extraction method based on magnetic particle technology. To improve the grain-quality traits of two rice varieties, we have designed and employed an efficient codominant functional marker system (including Wx, ALK, Chalk5, and fgr genes), in combination with genotyping based on automated capillary electrophoresis. Rice lines with simultaneous improvement at multiple loci were obtained and found to have superior grain quality and to be fragrant. The genotyping pipeline established in this study represents an efficient, reliable, and precise platform for MAS.     

KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposi''s sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi''s sarcoma, primary effusion lymphoma and multicentric Castleman''s disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.     

The ING4 Binding with p53 and Induced p53 Acetylation were Attenuated by Human Papillomavirus 16 E6

High risk subtype HPV16 early oncoprotein E6 contributes host cell immortalization and transformation through interacting with a number of cellular factors. ING4 is one member of the inhibitor of growth (ING) family of type II tumor suppressors and it has been shown to be involved in regulating p53 function. However, the effect and mechanism of HPV16 E6 on ING4 function remain elusive. In this study, we report HPV16 E6 combines with ING4 in vivo and in vitro. The ING4 induced p53 acetylation and its combining with p53 were attenuated by HPV16 E6 independent of p53 degradation. The enhancing function of ING4 on p53 mediated apoptosis was diminished when HPV16 E6 existed. These findings reveal that ING4 may be a common target of oncogenic viruses for driving host cell carcinogenesis.     

Expression pattern of NLRP3 and its related cytokines in the lung and brain of avian influenza virus H9N2 infected BALB/c mice

Background

H9N2 avian influenza virus (AIV) becomes the focus for its ability of transmission to mammals and as a donor to provide internal genes to form the new epidemic lethal influenza viruses. Residue 627 in PB2 has been proven the virulence factor of H9N2 avian influenza virus in mice, but the detailed data for inflammation difference between H9N2 virus strains with site 627 mutation is still unclear. The inflammasome NLRP3 is recently reported as the cellular machinery responsible for activation of inflammatory processes and plays an important role during the development of inflammation caused by influenza virus infection.

Methods

In this study, we investigated the expression pattern of NLRP3 and its related cytokines of IL-1β and TNF-α in BALB/c mice infected by H9N2 AIV strains with only a site 627 difference at both mRNA and protein levels at different time points.

Results

The results showed that the expression level of NLRP3, IL-1β and TNF-α changed in the lung and brain of BALB/c mice after infection by V_K627 and rV_K627E. The immunohistological results showed that the positive cells of NLRP3, IL-1β and TNF-α altered the positive levels of original cells in tissues and infiltrated inflammatory cells which caused by H9N2 infection.

Conclusions

Our results provided the basic data at differences in expression pattern of NLRP3 and its related cytokines in BALB/c mice infected by H9N2 influenza viruses with only a site 627 difference. This implied that NLRP3 inflammasome plays a role in host response to influenza virus infection and determines the outcome of clinical manifestation and pathological injury. This will explain the variable of pathological presentation in tissues and enhance research on inflammation process of the AIV H9N2 infection.

     

Regulation of myostatin expression is associated with growth and muscle development in commercial broiler and DMC muscle

Myostatin is a negative regulator of skeletal muscle growth. Muscle tissue is the largest tissue in the body and influences body growth. Commercial Avian broiler chickens are selected for high growth rate and muscularity. Daweishan mini chickens are a slow growing small-sized chicken breed. We investigated the relations between muscle (breast and leg) myostatin mRNA expression and body and muscle growth. Twenty chickens per breed were slaughtered at 0, 30, 60, 90, 120, and 150 days of age. Body and muscle weights were higher at all times in Avian chickens. Breast muscle myostatin expression was higher in Avian chickens than in Daweishan mini chickens at day 30. Myostatin expression peaked at day 60 in Daweishan mini chickens and expression remained higher in breast muscle. Daweishan mini chickens myostatin expression correlated positively with carcass weight, breast and leg muscle weight from day 0 to 60, and correlated negatively with body weight from day 90 to 150, while myostatin expression in Avian chickens was negatively correlated with carcass and muscle weight from day 90 to 150. The results suggest that myostatin expression is related to regulation of body growth and muscle development, with two different regulatory mechanisms that switch between days 30 and 60.     

Simultaneous removal of carbon and nitrogen from municipal-type synthetic wastewater using net-like rotating biological contactor (NRBC)

The treatment of municipal-type synthetic wastewater was carried out using a three stages net-like rotating biological contactor (NRBC). The results indicated that, compared with conventional rotating biological contactor (RBC), NRBC have several advantages, such as quick start-up, high biomass concentration and can handle high organic loading rates. The COD and total nitrogen removal rates achieved were 78.8–89.7% and 40.2–61.4%, respectively, in aerobic treatment of low COD municipal-type wastewater at hydraulic retention times (HRT) from 5 to 9 h. The COD removal rate achieved 80–95% when organic loading varied between 16 and 40 gCOD/m² d. A large amount of nematodes were found in the NRBC system, which made the NRBC system produce relatively low amounts of waste sludge, due to their grazing.     

Synaptic neurotransmission depression in ventral tegmental dopamine neurons and cannabinoid-associated addictive learning

Drug addiction is an association of compulsive drug use with long-term associative learning/memory. Multiple forms of learning/memory are primarily subserved by activity- or experience-dependent synaptic long-term potentiation (LTP) and long-term depression (LTD). Recent studies suggest LTP expression in locally activated glutamate synapses onto dopamine neurons (local Glu-DA synapses) of the midbrain ventral tegmental area (VTA) following a single or chronic exposure to many drugs of abuse, whereas a single exposure to cannabinoid did not significantly affect synaptic plasticity at these synapses. It is unknown whether chronic exposure of cannabis (marijuana or cannabinoids), the most commonly used illicit drug worldwide, induce LTP or LTD at these synapses. More importantly, whether such alterations in VTA synaptic plasticity causatively contribute to drug addictive behavior has not previously been addressed. Here we show in rats that chronic cannabinoid exposure activates VTA cannabinoid CB1 receptors to induce transient neurotransmission depression at VTA local Glu-DA synapses through activation of NMDA receptors and subsequent endocytosis of AMPA receptor GluR2 subunits. A GluR2-derived peptide blocks cannabinoid-induced VTA synaptic depression and conditioned place preference, i.e., learning to associate drug exposure with environmental cues. These data not only provide the first evidence, to our knowledge, that NMDA receptor-dependent synaptic depression at VTA dopamine circuitry requires GluR2 endocytosis, but also suggest an essential contribution of such synaptic depression to cannabinoid-associated addictive learning, in addition to pointing to novel pharmacological strategies for the treatment of cannabis addiction.     

Engineered heart tissue graft derived from somatic cell nuclear transferred embryonic stem cells improve myocardial performance in infarcted rat heart

The concept of regenerating diseased myocardium by implanting engineered heart tissue (EHT) is intriguing. Yet it was limited by immune rejection and difficulties to be generated at a size with contractile properties. Somatic cell nuclear transfer is proposed as a practical strategy for generating autologous histocompatible stem (nuclear transferred embryonic stem [NT‐ES]) cells to treat diseases. Nevertheless, it is controversial as NT‐ES cells may pose risks in their therapeutic application. EHT from NT‐ES cell‐derived cardiomyocytes was generated through a series of improved techniques in a self‐made mould to keep the EHTs from contraction and provide static stretch simultaneously. After 7 days of static and mechanical stretching, respectively, the EHTs were implanted to the infarcted rat heart. Four weeks after transplantation, the suitability of EHT in heart muscle repair after myocardial infarction was evaluated by histological examination, echocardiography and multielectrode array measurement. The results showed that large (thickness/diameter, 2–4 mm/10 mm) spontaneously contracting EHTs was generated successfully. The EHTs, which were derived from NT‐ES cells, inte grated and electrically coupled to host myocardium and exerted beneficial effects on the left ventricular function of infarcted rat heart. No teratoma formation was observed in the rat heart implanted with EHTs for 4 weeks. NT‐ES cells can be used as a source of seeding cells for cardiac tissue engineering. Large contractile EHT grafts can be constructed in vitro with the ability to survive after implantation and improve myocardial performance of infarcted rat hearts.     

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.