Establishment of macaque trophoblast stem cell lines derived from cynomolgus monkey blastocysts.

The placenta forms a maternal-fetal junction that supports many physiological functions such as the supply of nutrition and exchange of gases and wastes. Establishing an in vitro culture model of human and non-human primate trophoblast stem/progenitor cells is important for investigating the process of early placental development and trophoblast differentiation. In this study, we have established five trophoblast stem cell (TSC) lines from cynomolgus monkey blastocysts, named macTSC #1-5. Fibroblast growth factor 4 (FGF4) enhanced proliferation of macTSCs, while other exogenous factors were not required to maintain their undifferentiated state. macTSCs showed a trophoblastic gene expression profile and trophoblast-like DNA methylation status and also exhibited differentiation capacity towards invasive trophoblast cells and multinucleated syncytia. In a xenogeneic chimera assay, these stem cells contributed to trophectoderm (TE) development in the chimeric blastocysts. macTSC are the first primate trophoblast cell lines whose proliferation is promoted by FGF4. These cell lines provide a valuable in vitro culture model to analyze the similarities and differences in placental development between human and non-human primates.

A reduced M1-like/M2-like ratio of macrophages in healthy adipose tissue expansion during SGLT2 inhibition.

The adipose tissue includes various stromal cells, such as preadipocytes, endothelial cells, fibroblasts, and immune cells, which are involved in adipose tissue functions. We previously reported that, in obese mice, the sodium-glucose cotransporter 2 inhibitor ipragliflozin (Ipra) promoted the expansion of the epididymal adipose tissue (Epi) with increase of serum ketone body concentration. The Ipra-induced adipose tissue expansion did not deteriorate adipose inflammation, or systemic glucose/lipid metabolism, referred to as "healthy adipose tissue expansion." Here we found that Ipra promoted healthy adipose tissue expansion with a reduced ratio of pro-inflammatory M1-like adipose tissue macrophages (ATMs) to anti-inflammatory M2-like ATMs. Ipra downregulated the gene expression of interleukin (IL)-15 (Il15) in stromal cells of Epi. IL-15 inhibited lipogenesis in 3T3-L1 cells associated with downregulation of the lipogenic gene. Ketone body ß-hydroxybutyrate suppressed Il15 gene induction in M1-polarized cultured macrophages, and a ketogenic diet reproduced the adipose tissue expansion without deteriorating systemic glucose metabolism in mice. Our data indicate that the phenotypic switch of ATMs could mediate healthy adipose tissue expansion by treatment with Ipra, and it may offer new insights into the pathophysiological mechanisms of adipose tissue expansion.

Canagliflozin, an SGLT2 inhibitor, attenuates the development of hepatocellular carcinoma in a mouse model of human NASH.

Sodium glucose cotransporter 2 (SGLT2) inhibitors, an antidiabetic drug, promotes urinary excretion of glucose by blocking its reabsorption in the renal proximal tubules. It is unclear whether SGLT2 inhibition could attenuate nonalcoholic steatohepatitis (NASH) and NASH-associated hepatocellular carcinoma. We examined the preventive effects of an SGLT2 inhibitor canagliflozin (CANA) in Western diet (WD)-fed melanocortin 4 receptor-deficient (MC4R-KO) mice, a mouse model of human NASH. An eight-week CANA treatment attenuated hepatic steatosis in WD-fed MC4R-KO mice, with increased epididymal fat mass without inflammatory changes. CANA treatment for 20 weeks inhibited the development of hepatic fibrosis in WD-fed MC4R-KO mice. After one year of CANA treatment, the number of liver tumors was significantly reduced in WD-fed MC4R-KO mice. In adipose tissue, CANA suppressed the ratio of oxidative to reduced forms of glutathiones (GSSG/GSH) in WD-fed MC4R-KO mice. Treatment with GSH significantly attenuated the H2O2-induced upregulation of genes related to NADPH oxidase in 3T3-L1 adipocytes, and that of Il6, Tgfb, and Pdgfb in RAW264.7 cells. This study provides evidence that SGLT2 inhibitors represent the unique class of drugs that can attenuate or delay the onset of NASH and eventually hepatocellular carcinoma, at least partly, through "healthy adipose expansion".

Distinct clinicopathologic characteristics of lung mucinous adenocarcinoma with KRAS mutation.

Primary mucinous adenocarcinomas are uncommon, and their pathogenesis remains unclear. We recently reported the clinicopathologic characteristics of surgically resected mucinous adenocarcinoma, including the frequent involvement of the left and lower lung and absence of central fibrosis. The present study attempted to clarify the pathogenesis of mucinous adenocarcinoma based on KRAS mutation status. We selected 45 mucinous adenocarcinoma cases from among 2474 surgically resected primary lung adenocarcinomas. Of these, 22 had a KRAS mutation (48.9%), whereas only 7 (15.6%) had an epidermal growth factor receptor mutation, and 2 cases had both mutations. The mucinous adenocarcinomas with KRAS mutations were located in the lower lung lobe significantly more often (P < .05) than were tumors without KRAS mutation. The mucinous adenocarcinoma cases with KRAS mutations also had a significantly lower frequency of nuclear atypia (P < .05). We compared the degree of immunostaining for matrix metalloproteinase-7 (MMP-7), laminin-5, and geminin in the mucinous adenocarcinoma with and without KRAS mutation. The proportion of geminin-positive cells was lower among the cases with a mutation than among those without (0.7% versus 2.1%; P < .05). No significant differences in the extent of staining of the other markers were observed between the groups. The current study clearly demonstrated that mucinous adenocarcinomas with KRAS mutations have clinicopathologic characteristics different from those of mucinous adenocarcinoma without such mutations.

Lexical representation of Japanese vowel devoicing.

Vowel devoicing happens in Japanese when the high vowel is between voiceless consonants. The aim of this study is to investigate the lexical representation of vowel devoicing. A long-term repetition-priming experiment was conducted. Participants shadowed words containing either a devoiced or a voiced vowel in three priming paradigms, and their shadow responses were analyzed. It was found that participants produced the phonologically appropriate allophone most of the time based on the consonantal environments. Shadowing latencies for the voiced stimuli were faster than for the devoiced stimuli in the environment where the vowel should be voiced; while, no significant RT difference was observed between the two forms in the environment where vowel devoicing was expected. In addition, a priming effect between the devoiced and voiced stimuli emerged only in the devoicing environment. The results suggest that since vowel devoicing is very common in spoken Japanese, the devoiced form may be stored in the lexicon. The results also suggest a link between the two forms in the lexicon and a direct access between an input and a lexical representation without going through intermediate levels that usually cost extra processes.

Feasibility and robustness of amplification refractory mutation system (ARMS)-based KRAS testing using clinically available formalin-fixed, paraffin-embedded samples of colorectal cancers.

BACKGROUND: KRAS mutation testing is recommended for the discernment of metastatic colorectal cancer patients who are unlikely to benefit from anti-epidermal growth factor receptor antibodies. A recently developed amplification refractory mutation-Scorpion system is becoming a standard method for KRAS mutant detection. The feasibility and robustness of this system using DNA samples from clinically available formalin-fixed, paraffin-embedded specimens were evaluated. METHODS: Genomic DNA from macro-dissected 110 specimens was applied for the KRAS mutant detection using a commercial amplification refractory mutation-Scorpion system kit. Success rate and mutant detection rate of the test were evaluated. RESULTS: Small intra- and inter-lot deviations of the testing kit and a good concordance among different real-time polymerase chain reaction systems suggested the reliability of the amplification refractory mutation-Scorpion system. Though one-third of the 110 samples that were tested did not contain a sufficient amount of DNA to detect a 1% concentration of mutant alleles, the mutant detection rate was not impaired using tumor DNA concentrated by macro-dissection. Using a higher amount of template DNA, which supposedly contained abundant interfering substances, prevented the detection of the exogenous control amplicons, resulting in a reduced success rate. Adjusting the template amount according to the total DNA concentration might reduce the failure rate. CONCLUSION: The amplification refractory mutation-Scorpion system with formalin-fixed, paraffin-embedded specimen-derived DNA samples exhibited an acceptable feasibility and robustness suitable for routine clinical practice.

Biased discordance of KRAS mutation detection in archived colorectal cancer specimens between the ARMS-Scorpion method and direct sequencing.

OBJECTIVE: The concordance of KRAS mutation detection between the amplification refractory mutation system-Scorpion assay and direct sequencing was evaluated with clinically available formalin-fixed, paraffin-embedded specimens of metastatic colorectal cancers. METHODS: Genomic DNA from 120 macrodissected specimens was examined by the amplification refractory mutation system-Scorpion assay and direct sequencing. DNA mixtures of wild-type and mutant KRAS genes were prepared from the peripheral blood and the SW620 human colon cancer cell line for the model experiments. RESULTS: KRAS mutation was identified in 50 samples (41.7%) by the amplification refractory mutation system-Scorpion assay and 42 samples (35.0%) by direct sequencing. Discordance between the two methods was observed for samples with smaller amounts of amplifiable DNA. The sensitivity of direct sequencing was impaired by the decrease in template DNA and polymerase chain reaction cycles in the experimental models. CONCLUSIONS: Decreased sensitivity of direct sequencing caused by insufficient polymerase chain reaction amplification resulted in biased discordance between direct sequencing and amplification refractory mutation system-Scorpion. Polymerase chain reaction conditions satisfactory for amplifying tens of haploid copies of genomic DNA to the saturation level might be necessary to ensure the robustness of the direct sequencing-based method employed for formalin-fixed, paraffin-embedded specimen-derived DNA samples.

Sensitivity of breast cancer cell lines to the novel insulin-like growth factor-1 receptor (IGF-1R) inhibitor NVP-AEW541 is dependent on the level of IRS-1 expression.

To investigate the potential value of targeting insulin-like growth factor-1 receptor (IGF-1R) in breast cancer, we examined the effects of NVP-AEW541, a selective small-molecule inhibitor of the IGF-1R tyrosine kinase, in a panel of 16 breast cancer cell lines. All cell lines expressed IGF-1R, but MCF-7 expressed much higher levels of insulin receptor substrate-1 (IRS-1) than the others. NVP-AEW541 was more potent at inhibiting growth of MCF-7 cells as compared to the others (IC(50), 1 microM vs. approximately 7 microM). Comparing MCF-7 to T47D cells, which express IGF-1R at a level identical to MCF-7 but have less than 1/30 the amount of IRS-1, NVP-AEW541 caused cell-cycle arrest at the G1-S boundary, reduced in vitro cell migration, and enhanced the cytotoxic effects of vinorelbine and paclitaxel in MCF-7, but not in T47D. While NVP-AEW541 decreased the phosphorylation of IGF-1R in both cell lines, it inhibited phosphorylation of Akt and disrupted the IRS-1/PI3K complex only in MCF-7. These findings suggest that inhibiting IGF-1R may be an effective therapeutic strategy for breast cancers that co-express IGF-1R and IRS-1 at high levels.