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Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

•We produced NY-ESO-1 peptide/HLA-DR*08:03 tetramers.•We showed a discrepancy between free peptide recognition and tetramer binding.•NY-ESO-1 123–135/HLA-DR*08:03 tetramer detected ex vivo CD4 responses in patients. We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121–138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1 125–134 and 124–134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122–135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122–135 on APC, but the peptide 122–135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123–135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring.

Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64 Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that 64 Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64 Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18 F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87–100, DQB1*0401-restricted NY-ESO-1 95–107 and DRB1*0803-restricted NY-ESO-1 124–134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination.

The morphological features (i.e. descriptors beyond total length) of cetacean spermatozoa have been reported (albeit often minimally) for only 14 of the nearly 80 extant species. These 14 species represent six families, one from the suborder Mysticeti and five from the suborder Odontoceti (Matano et al. 1976; Fleming et al. 1981; Mogoe et al. 1998; Beilis et al. 2000; Kita et al. 2001; Miller et al. 2002; Fukui et al. 2004; Meisner et al. 2005). Two investigations have been comparative studies of cetacean spermatozoa, one employing light microscopy (LM) and scanning electron microscopy (SEM) (Kita et al. 2001) and the other using LM, SEM and transmission electron microscopy (TEM) (Miller et al. 2002). In addition, Meisner et al. (2005) included five cetaceans in their SEM examination of sperm head morphology of 36 mammalian species. Although most accounts have been of epididymal spermatozoa, Mogoe et al. (1998) described sperm from the vasa deferens. Others examined spermatozoa collected using electroejaculation (Fleming et al. 1981) or trained medical behaviors (Miller et al. 2002). Fukui et al. (2004) noted the occurrence of significant morphological changes secondary to cryopreservation. This chapter reviews the morphological and ultrastructural characteristics reported for cetacean spermatozoa and provides new information on spermatozoa from some species investigated previously, as well as from five other species, including representatives of two additional families. Lastly, we offer observations on the ultrastructural effects of cryopreservation.

We have conducted a NanoSIMS-based search for presolar material in samples recently returned from C-type asteroid Ryugu as part of JAXA's Hayabusa2 mission. We report the detection of all major presolar grain types with O- and C-anomalous isotopic compositions typically identified in carbonaceous chondrite meteorites: 1 silicate, 1 oxide, 1 O-anomalous supernova grain of ambiguous phase, 38 SiC, and 16 carbonaceous grains. At least two of the carbonaceous grains are presolar graphites, whereas several grains with moderate C isotopic anomalies are probably organics. The presolar silicate was located in a clast with a less altered lithology than the typical extensively aqueously altered Ryugu matrix. The matrix-normalized presolar grain abundances in Ryugu are 4.8−2.6+4.7 ppm for O-anomalous grains, 25−5+6 ppm for SiC grains, and 11−3+5 ppm for carbonaceous grains. Ryugu is isotopically and petrologically similar to carbonaceous Ivuna-type (CI) chondrites. To compare the in situ presolar grain abundances of Ryugu with CI chondrites, we also mapped Ivuna and Orgueil samples and found a total of 15 SiC grains and 6 carbonaceous grains. No O-anomalous grains were detected. The matrix-normalized presolar grain abundances in the CI chondrites are similar to those in Ryugu: 23−6+7 ppm SiC and 9.0−3.6+5.4 ppm carbonaceous grains. Thus, our results provide further evidence in support of the Ryugu–CI connection. They also reveal intriguing hints of small-scale heterogeneities in the Ryugu samples, such as locally distinct degrees of alteration that allowed the preservation of delicate presolar material.

The nucleosynthetic isotope composition of planetary materials provides a record of the heterogeneous distribution of stardust within the early solar system. In 2020 December, the Japan Aerospace Exploration Agency Hayabusa2 spacecraft returned to Earth the first samples of a primitive asteroid, namely, the Cb-type asteroid Ryugu. This provides a unique opportunity to explore the kinship between primitive asteroids and carbonaceous chondrites. We report high-precision μ 26Mg* and μ 25Mg values of Ryugu samples together with those of CI, CM, CV, and ungrouped carbonaceous chondrites. The stable Mg isotope composition of Ryugu aliquots defines μ 25Mg values ranging from –160 ± 20 ppm to –272 ± 30 ppm, which extends to lighter compositions relative to Ivuna-type (CI) and other carbonaceous chondrite groups. We interpret the μ 25Mg variability as reflecting heterogeneous sampling of a carbonate phase hosting isotopically light Mg (μ 25Mg ∼ –1400 ppm) formed by low temperature equilibrium processes. After correcting for this effect, Ryugu samples return homogeneous μ 26Mg* values corresponding to a weighted mean of 7.1 ± 0.8 ppm. Thus, Ryugu defines a μ 26Mg* excess relative to the CI and CR chondrite reservoirs corresponding to 3.8 ± 1.1 and 11.9 ± 0.8 ppm, respectively. These variations cannot be accounted for by in situ decay of 26Al given their respective 27Al/24Mg ratios. Instead, it requires that Ryugu and the CI and CR parent bodies formed from material with a different initial 26Al/27Al ratio or that they are sourced from material with distinct Mg isotope compositions. Thus, our new Mg isotope data challenge the notion that Ryugu and CI chondrites share a common nucleosynthetic heritage.

Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. However, the molecular link between obesity and ER stress remains to be identified. Proteasomes are important multicatalytic enzyme complexes that degrade misfolded and oxidized proteins. Here, we report that both mouse models of obesity and diabetes and proteasome activator (PA)28-null mice showed 30–40% reduction in proteasome activity and accumulation of polyubiquitinated proteins in the liver. PA28-null mice also showed hepatic steatosis, decreased hepatic insulin signaling, and increased hepatic glucose production. The link between proteasome dysfunction and hepatic insulin resistance involves ER stress leading to hyperactivation of c-Jun NH2-terminal kinase in the liver. Administration of a chemical chaperone, phenylbutyric acid (PBA), partially rescued the phenotypes of PA28-null mice. To confirm part of the results obtained from in vivo experiments, we pretreated rat hepatoma-derived H4IIEC3 cells with bortezomib, a selective inhibitor of the 26S proteasome. Bortezomib causes ER stress and insulin resistance in vitro—responses that are partly blocked by PBA. Taken together, our data suggest that proteasome dysfunction mediates obesity-induced ER stress, leading to insulin resistance in the liver.

Rock fragments of the Cb-type asteroid Ryugu returned to Earth by the JAXA Hayabusa2 mission share mineralogical, chemical, and isotopic properties with the Ivuna-type (CI) carbonaceous chondrites. Similar to CI chondrites, these fragments underwent extensive aqueous alteration and consist predominantly of hydrous minerals likely formed in the presence of liquid water on the Ryugu parent asteroid. Here we present an in situ analytical survey performed by secondary ion mass spectrometry from which we have estimated the D/H ratio of Ryugu’s hydrous minerals, D/HRyugu, to be [165 ± 19] × 10−6, which corresponds to δDRyugu = +59 ± 121‰ (2σ). The hydrous mineral D/HRyugu’s values for the two sampling sites on Ryugu are similar; they are also similar to the estimated D/H ratio of hydrous minerals in the CI chondrites Orgueil and Alais. This result reinforces a link between Ryugu and CI chondrites and an inference that Ryugu’s samples, which avoided terrestrial contamination, are our best proxy to estimate the composition of water at the origin of hydrous minerals in CI-like material. Based on this data and recent literature studies, the contribution of CI chondrites to the hydrogen of Earth’s surficial reservoirs is evaluated to be ∼3%. We conclude that the water responsible for the alteration of Ryugu’s rocks was derived from water ice precursors inherited from the interstellar medium; the ice partially re-equilibrated its hydrogen with the nebular H2 before being accreted on the Ryugu’s parent asteroid.