Emulsified Phosphatidylserine,Simple and Effective Peptide Carrier for Induction of Potent Epitope-Specific T Cell Responses

Background

To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) in vivo. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability.

Methodology/Principal Findings

Data from an epitope-specific in vivo CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c⁺CD11b⁺MHCII⁺ conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells in vivo.

Conclusions/Significance

This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses in vivo. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.     

Environmental DNA as a ‘Snapshot’ of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay,Sea of Japan

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R² value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.     

The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand) signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinase 3β) inhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.     

Combining mapping of physiological quantitative trait loci and transcriptome for cold tolerance for counteracting male sterility induced by low temperatures during reproductive stage in rice

Male sterility induced by low temperatures (LTs) during the reproductive stage is a major constraint for temperate zone rice. To detect physiological quantitative trait loci (QTLs), we modeled genotypic variation in the physiological processes involved in low temperature spikelet sterility on the basis of anther length (AL), a proxy for microspore and pollen grain number per anther. The model accounted for 83% of the genotypic variation in potential AL at normal temperature and the ability to maintain AL at LT. We tested the model on 208 recombinant inbred lines of cold‐tolerant ‘Tohoku‐PL3’ (PL3) × cold‐sensitive ‘Akihikari’ (AH) for 2 years. QTLs for spikelet fertility (FRT) at LT were detected on chromosomes 5 (QTL for Cold Tolerance at Reproductive stage, qCTR5) and 12 (qCTR12). qCTR12 was annotated with the ability to maintain AL under LTs. qCTR5 was in a region shared with QTLs for culm length and heading date. Genome‐wide expression analysis showed 798 genes differentially expressed in the spikelets between the parents at LTs. Of these, 12 were near qCTR5 and 23 were near qCTR12. Gene expression analysis confirmed two candidate genes for qCTR5 (O‐methyltransferase ZRP4, Os05g0515600; beta‐1,3‐glucanase‐like protein, Os05g0535100) and one for qCTR12 (conserved hypothetical protein, Os12g0550600). Nucleotide polymorphisms (21 deletions, 2 insertions and 10 single nucleotide polymorphisms) in PL3 were found near the candidate conserved hypothetical protein (Os12g0550600) and upstream in PL3, but not in AH. Haplotype analysis revealed that this gene came from ‘Kuchum’. The combination of mapping physiological QTLs with gene expression analysis can be extended to identify other genes for abiotic stress response in cereals.     

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.     

Sequential Ubiquitination of Ribosomal Protein uS3 Triggers the Degradation of Non-functional 18S rRNA

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Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth

Cyclic AMP regulates multiple neuronal functions, including neurite outgrowth and axonal regeneration. GPR3, GPR6, and GPR12 make up a family of constitutively active G protein-coupled receptors (GPCRs) that share greater than 50% identity and 65% similarity at the amino acid level. They are highly expressed in the central nervous system, and their expression in various cell lines results in constitutive stimulation of cAMP production. When the constitutively active GPCRs were overexpressed in rat cerebellar granule neurons in culture, the transfected neurons exhibited significantly enhanced neurite outgrowth and overcame growth inhibition caused by myelin-associated glycoprotein. GPR12-mediated neurite outgrowth was the most prominent and was shown to depend on G(s) and cAMP-dependent protein kinase. Moreover, the GPR12-mediated rescue from myelin-associated glycoprotein inhibition was attributable to cAMP-dependent protein kinase-mediated inhibition of the small GTPase, RhoA. Among the three receptors, GPR3 was revealed to be enriched in the developing rat cerebellar granule neurons. When the endogenous GPR3 was knocked down, significant reduction of neurite growth was observed, which was reversed by expression of either GPR3 or GPR12. Taken together, our results indicate that expression of the constitutively active GPCRs up-regulates cAMP production in neurons, stimulates neurite outgrowth, and counteracts myelin inhibition. Further characterization of the GPCRs in developing and injured mammalian neurons should provide insights into how basal cAMP levels are regulated in neurons and could establish a firm scientific foundation for applying receptor biology to treatment of various neurological disorders.     

Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.