We have built a digital nuclear atlas of the newly hatched, first larval stage (L1) of the wild type hermaphrodite of C. elegans at single cell resolution from confocal image stacks of 15 individuals. The atlas quantifies the stereotypy of the locations and provides for other statistics on the spatial patterns of the 357 nuclei that could be faithfully segmented and annotated of the 558 present at this developmental stage. Given this atlas we then developed an automated approach to assign cell names to each nucleus in a 3D image of an L1 worm. We achieve 86% accuracy in identifying the 357 nuclei automatically. This computational method is essential for high-throughput single cell analyses of the worm at post-embryonic stages, such as determining the expression of every gene in every cell during development from the L1 onward, or ablating or stimulating cells under computer control in a high-throughput functional screen.

Many studies have shown that tumor‐associated macrophages (TAMs) contribute to tumor development and poor prognosis in various cancers. In this study, we investigated the macrophage populations and phenotypes, and their correlation to angiogenesis, immunosuppression, and clinical prognosis in intrahepatic cholangiocarcinoma (ICC). CD68 (+) and CD163 (+) macrophage infiltration was analyzed in paraffin‐embedded tissue samples from 39 patients. CD163 is used as a marker of M2 macrophages. Neovascularization and infiltration of forkhead box P3 (FOXP3) (+) regulatory T cells were also evaluated. The number of CD68 (+) and CD163 (+) macrophages was positively correlated with the numbers of vessels and regulatory T cells. The number of CD163 (+) cells was more closely associated with them. Intrahepatic cholangiocarcinoma (ICC) patients with high counts of CD163 (+) macrophages showed poor disease‐free survival (P = 0.0426). The macrophage density was not correlated with overall survival. In an in vitro study using ICC cell lines (HuCCT1, RBE, and MEC) and human macrophages, tumor cell supernatant (TCS) from cell lines induced an activation of signal transducers and activators of transcription‐3 (Stat3) and macrophage polarization toward the M2 phenotype. Tumor cell supernatant (TCS) from HuCCT1 most strongly induced Stat3 activation and production of cytokines and other bioactive molecules such as interleukin (IL)‐10, vascular endothelial growth factor (VEGF)‐A, transforming growth factor (TGF)‐β, and matrix metalloproteinase (MMP)‐2. Down‐regulation of Stat3 by siRNA significantly suppressed the production of IL‐10 and VEGF‐A. These results provide suggestive evidence that TAMs contribute to cancer progression via Stat3 activation, and CD163 is useful for evaluating M2 TAMs and predicting the clinical prognosis of ICC patients. (Cancer Sci)

Highlights d CAD, the rate-limiting de novo pyrimidine synthesis enzyme, deamidates RelA d Deamidation shunts RelA from mediating an inflammatory response to aerobic glycolysis d Deamidation of RelA occurs in a cell cycle-dependent manner d Cancer cells hijack RelA deamidation to promote glycolysis and tumorigenesis

Glutamate decarboxylase (GAD, EC 4.1.1.15) has been suggested to be a key, regulatory point in the biosynthesis of γ-aminobutyrate (GABA) and in the utilization of citric acid through GABA shunt pathway. In this study we discovered two GAD genes, named as CsGAD1 and CsGAD2, in citrus genome database and then successfully cloned. Both CsGAD1 and CsGAD2 have a putative pyridoxal 5-phosphate binding domain in the middle region and a putative calmodulin-binding domain at the carboxyl terminus. Gene structure analysis showed that much difference exists in the size of exons and introns or in cis-regulatory elements in promoter region between the two GAD genes. Gene expression indicated that CsGAD1 transcript was predominantly expressed in flower and CsGAD2 transcript was predominantly expressed in fruit juice sacs; in the ripening fruit, CsGAD1 transcript level was at least 2-time higher than CsGAD2 transcript level. Moreover, CsGAD1 transcript level was increased significantly along with the increase of GAD activity and accompanied by a significant decrease of titratable acid (TA), suggesting that it is CsGAD1 rather than CsGAD2 plays a role in the citric acid utilization during fruit ripening. In addition, injection of abscisic acid and foliar spray of K2SO4 significantly increased the TA content of Satsuma mandarin, and significantly decreased GAD activity as well as CsGAD1 transcript, further suggesting the important role of CsGAD1 in the citrate utilization of citrus fruit.

No abstract

Class A scavenger receptors (SR‐A, CD204) are highly expressed in tumor‐associated macrophages (TAM). To investigate the function of SR‐A in TAM, wild‐type and SR‐A‐deficient (SR‐A−/−) mice were injected with EL4 cells. Although these groups of mice did not differ in the numbers of infiltrating macrophages and lymphocytes and in neovascularization, SR‐A−/− mice had delayed growth of EL4 tumors. Expression of inducible nitric oxide (NO) synthase and interferon (IFN)‐γ mRNA increased significantly in tumor tissues from SR‐A−/− mice. Engulfment of necrotic EL4 cells induced upregulation of NO and IFN‐γ production by cultured macrophages, and production of NO and IFN‐γ increased in SR‐A−/− macrophages in vitro. IFN‐β production by cultured macrophages was also elevated in SR‐A−/− macrophages in vitro. These results suggested that the antitumor activity of macrophages increased in SR‐A−/− mice because of upregulation of NO and IFN‐γ production. These data indicate an important role of SR‐A in regulating TAM function by inhibiting toll‐like receptor (TLR)4–IFN‐β signaling. (Cancer Sci 2009); 00: 000–000)

No abstract

Background/Aims The broadly antiviral cytokine interferon-α (IFNα)’s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN’s antiviral effects. We hypothesized that SART1 regulates antiviral effector genes (IEGs) through mRNA processing and splicing. Methods We performed siRNA knockdown in Huh7.5.1 cells and mRNA-seq with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. Results we identified 419 genes with greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical ISG mRNA transcription including MX1 and OAS3. However, SART1 did not affect Jak-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. Conclusions The spliceosome factor SART1 is not IFN-inducible but is an IFN effector gene. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have effects on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.