Specific Targeting of a Naturally Presented Osteosarcoma Antigen,Papillomavirus Binding Factor Peptide,Using an Artificial Monoclonal Antibody

Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (K_D = 1.53 × 10⁻⁹ m) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy.     

Molecular evolution of VP3, VP1, 3Cpro and 3Dpol coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa,Japan

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C^pro and 3D^pol coding regions performed. To assess time‐scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p‐distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10^?3 substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77–0.94. The p‐distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

     

Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris

We developed a method for efficient chromosome tagging in Pichia pastoris, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from P. pastoris. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.     

ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

In the skin lesions of atopic dermatitis (AD), keratinocytes release large quantities of thymic stromal lymphopoietin (TSLP), causing unfavorable inflammation along with skin damage. Nevertheless, how TSLP influences keratinocytes themselves is still unknown. In this study, we showed that ΔNp63, a p53-homologue, predominantly expressed in keratinocytes regulated the receptor complex of TSLP, which determines susceptibility to self-derived TSLP. Expression of TSLP receptors in skin tissues and keratinocytes was assessed by immunohistochemistry and quantitative RT-PCR, and in vitro studies were also performed to examine the functional relevance of ΔNp63 in the expression of TSLP receptors and the constituting autocrine and/or paracrine pathway of TSLP under the condition of stimuli to innate receptors sensing cell damage. The results showed that normal keratinocytes in the upper epidermis preferentially expressed TSLP receptors and conversely lacked ΔNp63, which has an inhibitory effect on the expression of TSLP receptors. Interestingly, the epidermis of AD lesions was found to abundantly contain keratinocytes with low or undetectable levels of ΔNp63 (ΔNp63^lo/-). Moreover, in the absence of ΔNp63, keratinocytes readily presented TSLP and other cytokines by stimuli through Toll-like receptor 3 (TLR3). Together with the evidence that extrinsic TSLP itself augments TSLP production by keratinocytes without ΔNp63, the results indicate that ΔNp63^lo/- keratinocytes generate TSLP through a putative autocrine and/or paracrine pathway upon TLR3 stimulation within AD lesions, since moieties of damaged cells and pathogens stimulate TLR3.     

Fomiroid A,a Novel Compound from the Mushroom Fomitopsis nigra,Inhibits NPC1L1-Mediated Cholesterol Uptake via a Mode of Action Distinct from That of Ezetimibe

Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C₃₀H₄₈O₃. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe.     

Adaptive Iterative Dose Reduction Using Three Dimensional Processing (AIDR3D) Improves Chest CT Image Quality and Reduces Radiation Exposure

Objective

To assess the advantages of Adaptive Iterative Dose Reduction using Three Dimensional Processing (AIDR3D) for image quality improvement and dose reduction for chest computed tomography (CT).

Methods

Institutional Review Boards approved this study and informed consent was obtained. Eighty-eight subjects underwent chest CT at five institutions using identical scanners and protocols. During a single visit, each subject was scanned using different tube currents: 240, 120, and 60 mA. Scan data were converted to images using AIDR3D and a conventional reconstruction mode (without AIDR3D). Using a 5-point scale from 1 (non-diagnostic) to 5 (excellent), three blinded observers independently evaluated image quality for three lung zones, four patterns of lung disease (nodule/mass, emphysema, bronchiolitis, and diffuse lung disease), and three mediastinal measurements (small structure visibility, streak artifacts, and shoulder artifacts). Differences in these scores were assessed by Scheffe''s test.

Results

At each tube current, scans using AIDR3D had higher scores than those without AIDR3D, which were significant for lung zones (p<0.0001) and all mediastinal measurements (p<0.01). For lung diseases, significant improvements with AIDR3D were frequently observed at 120 and 60 mA. Scans with AIDR3D at 120 mA had significantly higher scores than those without AIDR3D at 240 mA for lung zones and mediastinal streak artifacts (p<0.0001), and slightly higher or equal scores for all other measurements. Scans with AIDR3D at 60 mA were also judged superior or equivalent to those without AIDR3D at 120 mA.

Conclusion

For chest CT, AIDR3D provides better image quality and can reduce radiation exposure by 50%.     

A Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates Parkin Recruitment

Parkinsonism typified by sporadic Parkinson disease is a prevalent neurodegenerative disease. Mutations in PINK1 (PTEN-induced putative kinase 1), a mitochondrial Ser/Thr protein kinase, or PARKIN, a ubiquitin-protein ligase, cause familial parkinsonism. The accumulation and autophosphorylation of PINK1 on damaged mitochondria results in the recruitment of Parkin, which ultimately triggers quarantine and/or degradation of the damaged mitochondria by the proteasome and autophagy. However, the molecular mechanism of PINK1 in dissipation of the mitochondrial membrane potential (ΔΨm) has not been fully elucidated. Here we show by fluorescence-based techniques that the PINK1 complex formed following a decrease in ΔΨm is composed of two PINK1 molecules and is correlated with intermolecular phosphorylation of PINK1. Disruption of complex formation by the PINK1 S402A mutation weakened Parkin recruitment onto depolarized mitochondria. The most disease-relevant mutations of PINK1 inhibit the complex formation. Taken together, these results suggest that formation of the complex containing dyadic PINK1 is an important step for Parkin recruitment onto damaged mitochondria.     

Parkin-catalyzed Ubiquitin-Ester Transfer Is Triggered by PINK1-dependent Phosphorylation

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.     

Reproductive interference hampers species coexistence despite conspecific sperm precedence

Negative interspecific mating interactions, known as reproductive interference, can hamper species coexistence in a local patch and promote niche partitioning or geographical segregation of closely related species. Conspecific sperm precedence (CSP), which occurs when females that have mated with both conspecific and heterospecific males preferentially use conspecific sperm for fertilization, might contribute to species coexistence by mitigating the costs of interspecific mating and hybridization. We discussed whether two species exhibiting CSP can coexist in a local environment in the presence of reproductive interference. First, using a behaviorally explicit mathematical model, we demonstrated that two species characterized by negative mating interactions are unlikely to coexist because the costs of reproductive interference, such as loss of mating opportunity with conspecific partners, are inevitably incurred when individuals of both species are present. Second, we experimentally examined differences in mating activity and preference in two Harmonia ladybird species known to exhibit CSP. These behavioral differences may lead to local extinction of H. yedoensis because of reproductive interference by H. axyridis. This prediction is consistent with field observations that H. axyridis uses various food sources and habitats whereas H. yedoensis is confined to a less preferred prey item and a pine tree habitat. Finally, by a comparative approach, we observed that niche partitioning or parapatric distribution, but not sympatric coexistence in the same habitat, is maintained between species with CSP belonging to a wide range of taxa, including vertebrates and invertebrates living in aquatic or terrestrial environments. Taken together, it is possible that reproductive interference may destabilize local coexistence even in closely related species that exhibit CSP.