Development of CD4+ macrophages from intrathymic T cell progenitors is induced by thymic epithelial cells

It was recently demonstrated that there are CD4(+) macrophages, which exhibit strong phagocytic activity, in the thymus. They are suggested to play an important role for the elimination of apoptotic thymocytes. However, the origin and nature of CD4(+) macrophages in the thymus remain unexplored. In this study, we describe that the most immature intrathymic progenitors (CD25(-)/CD44(+)/FcR(+)) give rise to CD4(+) macrophages by oncostatin M-responsive thymic epithelial cells (ORTEC) in an IL-7-dependent manner. Neither conditioned medium of ORTEC nor a mixture of cytokines induced CD4(+) macrophages, and oncostatin M receptor was not expressed in thymocytes, suggesting that the development of CD4(+) macrophages from the immature thymocytes requires a direct interaction with ORTEC. These results collectively suggest that the development of CD4(+) macrophages from the intrathymic T cell progenitors is induced by thymic epithelial cells.     

Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5′ Capped 2′-O Unmethylated RNA

The interferon-inducible protein with tetratricopeptide (IFIT) family proteins inhibit replication of some viruses by recognizing several types of RNAs, including 5′-triphosphate RNA and 5′ capped 2′-O unmethylated mRNA. However, it remains unclear how IFITs inhibit replication of some viruses through recognition of RNA. Here, we analyzed the mechanisms by which Ifit1 exerts antiviral responses. Replication of a Japanese encephalitis virus (JEV) 2′-O methyltransferase (MTase) mutant was markedly enhanced in mouse embryonic fibroblasts and macrophages lacking Ifit1. Ifit1 bound 5′-triphosphate RNA but more preferentially associated with 5′ capped 2′-O unmethylated mRNA. Ifit1 inhibited the translation of mRNA and thereby restricted the replication of JEV mutated in 2′-O MTase. Thus, Ifit1 inhibits replication of MTase-defective JEV by inhibiting mRNA translation through direct binding to mRNA 5′ structures.     

Prickle2 is localized in the postsynaptic density and interacts with PSD-95 and NMDA receptors in the brain

The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.     

Real-time PCR-based analysis of the human bile microRNAome identifies miR-9 as a potential diagnostic biomarker for biliary tract cancer

Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC.     

Paleosalinity in a brackish lake during the Holocene based on stable oxygen and carbon isotopes of shell carbonate in Nakaumi Lagoon, southwest Japan

Based on the relationship between salinity and δ¹⁸O and δ¹³C of modern shells in the Lake Nakaumi-Shinji lagoon system (southwestern Japan), where the salinity changes regularly from ca. 1 PSU to 34 PSU, a paleosalinity record for Nakaumi Lagoon during the Holocene has been derived from bulk mollusk shell δ¹⁸O and δ¹³C data. The robust relationships between the salinity and modern shell δ¹⁸O_ar and δ¹³C_ar (aragonite) were used to calibrate the paleosalinity reconstruction. The salinity relationships are expressed by the regressions:

Salinity (PSU)=3.86 δ¹⁸O_ar(‰ VPDB)+33.9 (n=18, r=0.978)     

Simultaneous determination of urinary dialkylphosphate metabolites of organophosphorus pesticides using gas chromatography-mass spectrometry

In this study, we developed a safe and sensitive method for the simultaneous determination of urinary dialkylphosphates (DAPs), metabolites of organophosphorus insecticides (OPs), including dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP), using a pentafluorobenzylbromide (PFBBr) derivatization and gas chromatography-mass spectrometry (GC-MS). Several parameters were investigated: pH on evaporation, reaction temperature and time for the derivatization, the use of an antioxidant for preventing oxidation, and a clean-up step. The pH was set at 6, adjusted with K2CO3, and the reaction temperature and time of derivatization were 80 degrees C and 30 min, respectively. Sodium disulfite was chosen as an antioxidant. The clean-up step was performed with a Florisil/PSE mini-column to remove the unreacted PFBBr and sample matrix. This established procedure markedly shortened the sample preparation time to only about 3 h, and completely inhibited the unwanted oxidization of dialkylthiophosphates. The limits of determination (LOD) were approximately 0.3 microg/L for DMP, and 0.1 microg/L for DEP, DMTP, and DETP in 5 mL of human urine. Within-series and between-day imprecision for the present method using pooled urine spiked with DAPs was less than 20.6% in the calibration range of 1-300 microg/L, and the mean recovery was 56.7-60.5% for DMP, 78.5-82.7% for DEP, 88.3-103.9% for DMTP, and 84.2-92.4% for DETP. This method detected geometric mean values of the urinary DAPs in Japanese with and without occupational exposure to OPs, 16.6 or 27.4 for DMP, 1.0 or 0.7 for DEP, 1.3 or 2.3 for DMTP, and 1.0 or 1.1 microg/L for DETP, respectively. The present method, which does not require special equipment except for GC-MS, is quick, safe, and sensitive enough to be adopted in routine biological monitoring of non-occupational as well as occupational exposure to OPs.