Sensing of individual stalled 80S ribosomes by Fap1 for nonfunctional rRNA turnover.

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.

Perceptual consequences of native and non-native clear speech.

Native talkers are able to enhance acoustic characteristics of their speech in a speaking style known as "clear speech," which is better understood by listeners than "plain speech." However, despite substantial research in the area of clear speech, it is less clear whether non-native talkers of various proficiency levels are able to adopt a clear speaking style and if so, whether this style has perceptual benefits for native listeners. In the present study, native English listeners evaluated plain and clear speech produced by three groups: native English talkers, non-native talkers with lower proficiency, and non-native talkers with higher proficiency. Listeners completed a transcription task (i.e., an objective measure of the speech intelligibility). We investigated intelligibility as a function of language background and proficiency and also investigated the acoustic modifications that are associated with these perceptual benefits. The results of the study suggest that both native and non-native talkers modulate their speech when asked to adopt a clear speaking style, but that the size of the acoustic modifications, as well as consequences of this speaking style for perception differ as a function of language background and language proficiency.

Bacterial keratoconjunctivitis caused by Staphylococcus argenteus belonging to sequence type 1223 isolated in Japan.

Staphylococcus argenteus, characterized by the formation of non-pigmented (white) colonies, was recently identified as a new lineage separated from Staphylococcus aureus. However, correct identification of this lineage is difficult because of the similar characteristics to S. aureus. Here, we describe the first known case of keratoconjunctivitis due to S. argenteus in a 64-year-old man with diabetes. The symptoms of the patient were not improved by antibiotic therapy using levofloxacin eye drops (15 mg/mL). The conjunctival scraping was cultured, and coagulase-positive staphylococci forming white colonies were detected. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry confirmed the species as S. argenteus with a spectral score of 1.97. After the antibiotic was changed to vancomycin eye drops (10 mg/mL), the patient's symptom clearly improved. Multi-locus sequence typing showed that this isolate belonged to sequence type 1223, which has been predominantly isolated worldwide. Furthermore, this isolate harbored various virulence genes associated with S. aureus, such as staphylococcal enterotoxins and leukocidin. Since only limited information is available for this organism, further studies are needed to establish the epidemiology of S. argenteus.

Dietary glucosylceramides suppress tumor growth in a mouse xenograft model of head and neck squamous cell carcinoma by the inhibition of angiogenesis through an increase in ceramide.

BACKGROUND: We previously reported that dietary glucosylceramides show cancer-prevention activity in a mouse xenograft model of human head and neck cancer cells (SCCKN). However, the mechanism was unclear. Ceramides, metabolites of glucosylceramides, induce apoptotic cell death in various malignancies. Here, we investigated the inhibitory effects of dietary glucosylceramides on tumor growth in vivo and in vitro. METHODS: SCCKN were subcutaneously inoculated into the right flanks of NOD/SCID mice. Mice were treated with or without dietary glucosylceramides (300 mg/kg) daily for 14 consecutive days after confirmation of tumor progression. Microvessel areas around the tumor were assessed by hematoxylin-eosin staining and immunohistochemistry of CD31, and, as markers for angiogenesis, protein levels of VEGF, VEGF receptor-2, and HIF-1α were assessed by Western blotting. Mass spectrometry was performed to measure the levels of sphingolipids in mouse serum after treatment with dietary glucosylceramides. RESULTS: Oral administration of glucosylceramides significantly decreased SCCKN growth in the xenograft model with inhibition of angioinvasion. In tumor-invasive areas, VEGF and HIF-1α in the tumor cells, and VEGF receptor-2 in endothelial cells decreased after treatment with dietary glucosylceramides. Dietary glucosylceramides increased serum levels of sphingosine-based ceramides as compared to the control. In SCCKN and UV♀2 cells, C6-ceramide suppressed the expressions of VEGF, VEGF receptor-2, and HIF-1α in vitro. CONCLUSION: These results suggest that dietary glucosylceramides trigger the de novo pathway of ceramide synthesis, indicating that sphingosine-based ceramide suppresses the growth of head and neck tumors through the inhibition of pro-angiogenic signals such as VEGF, VEGF receptor-2, and HIF-1α.

Checkpoint kinase inhibitor AZD7762 overcomes cisplatin resistance in clear cell carcinoma of the ovary.

OBJECTIVE: Checkpoint kinase (Chk) inhibitors are thought to increase the cytotoxic effects of DNA-damaging agents and are undergoing clinical trials. The present study was aimed to assess the potential to use the Chk1 and Chk2 inhibitor, AZD7762, with other anticancer agents in chemotherapy to treat ovarian clear cell carcinoma. METHODS: Four ovarian clear cell carcinoma cell lines were used in this study. We treated the cells with AZD7762 and anticancer agents, then assessed cell viability, cell cycle distribution, apoptosis, and the expression of protein in apoptotic pathways and molecules downstream of the Chk signaling pathways. We also investigated the effects of these drug combinations on tumor growth in a nude mouse xenograft model. RESULTS: Synergistic effects from the combination of AZD7762 and cisplatin were observed in all 4 cell lines. However, we observed additive effects when AZD7762 was combined with paclitaxel on all cell lines tested. AZD7762 effectively suppressed the Chk signaling pathways activated by cisplatin, dramatically enhanced expression of phosphorylated H2A.X, cleaved caspase 9 and PARP, decreased the proportion of cells in the gap 0/ gap 1 phase and the synthesis-phase fraction, and increased apoptotic cells. Combinations of small interfering RNA against Chk 1 and small interfering RNA against Chk2 enhanced the cytotoxic effect of cisplatin in both RMG-I and KK cells. Finally, treating mice-bearing RMG-I with AZD7762 and cisplatin significantly suppressed growth of tumors in a xenograft model. CONCLUSIONS: The present study indicates that chemotherapy with AZD7762 and cisplatin should be explored as a treatment modality for women with ovarian clear cell carcinoma.

The Effects of Acoustic and Semantic Enhancements on Perception of Native and Non-Native Speech.

Previous research has shown that native listeners benefit from clearly produced speech, as well as from predictable semantic context when these enhancements are delivered in native speech. However, it is unclear whether native listeners benefit from acoustic and semantic enhancements differently when listening to other varieties of speech, including non-native speech. The current study examines to what extent native English listeners benefit from acoustic and semantic cues present in native and non-native English speech. Native English listeners transcribed sentence final words that were of different levels of semantic predictability, produced in plain- or clear-speaking styles by Native English talkers and by native Mandarin talkers of higher- and lower-proficiency in English. The perception results demonstrated that listeners benefited from semantic cues in higher- and lower-proficiency talkers' speech (i.e., transcribed speech more accurately), but not from acoustic cues, even though higher-proficiency talkers did make substantial acoustic enhancements from plain to clear speech. The current results suggest that native listeners benefit more robustly from semantic cues than from acoustic cues when those cues are embedded in non-native speech.

Speaking rate normalization across different talkers in the perception of Japanese stop and vowel length contrasts.

Perception of duration is critically influenced by the speaking rate of the surrounding context. However, to what extent this speaking rate normalization is talker-specific is understudied. This experiment investigated whether Japanese listeners' perception of temporally contrastive phonemes is influenced by the speaking rate of the surrounding context, and more importantly, whether the effect of the contextual speaking rate persists across different talkers for different types of contrasts: a singleton-geminate stop contrast and short-long vowel contrast in Japanese. The results suggest that listeners generalized their rate-based adjustments to different talkers' speech regardless of whether the target contrasts depended on silent closure duration or vowel duration. Our results thus support the view that speaking rate normalization is an obligatory process that happens in the early phase of perception.

Discrete palladium clusters that consist of two mutually bisecting perpendicular planes.

The construction of novel molecules with unprecedented alignments of the constituent elements has revolutionized the field of functional materials. The arrangement of two or more planar subunits in a mutually perpendicular fashion is a frequently encountered approach to produce novel functional materials. Previous examples of such materials can be categorized into two well-investigated families: spiro-conjugated and dumbbell-shaped structures, wherein the two planes are aligned orthogonally via a single atom or an axis, respectively. This article describes a third family: reaction of [Pd(CN t Bu)2]3 with Sn3Me8 or Ge6Me12 afforded a Pd7Sn4 cluster and a Pd8Ge6 cluster that consist of two mutually bisecting perpendicular planes. In the Pd7Sn4 cluster, the two equivalent Pd5Sn2 planes share three palladium atoms that include a dihedral angle of 85.6°.

Sequential Ubiquitination of Ribosomal Protein uS3 Triggers the Degradation of Non-functional 18S rRNA.

18S non-functional rRNA decay (NRD) eliminates non-functional 18S rRNA with deleterious mutations in the decoding center. Dissociation of the non-functional 80S ribosome into 40S and 60S subunits is a prerequisite step for degradation of the non-functional 18S rRNA. However, the mechanisms by which the non-functional ribosome is recognized and dissociated into subunits remain elusive. Here, we report that the sequential ubiquitination of non-functional ribosomes is crucial for subunit dissociation. 18S NRD requires Mag2-mediated monoubiquitination followed by Hel2- and Rsp5-mediated K63-linked polyubiquitination of uS3 at the 212th lysine residue. Determination of the aberrant 18S rRNA levels in sucrose gradient fractions revealed that the subunit dissociation of stalled ribosomes requires sequential ubiquitination of uS3 by E3 ligases and ATPase activity of Slh1 (Rqt2), as well as Asc1 and Dom34. We propose that sequential uS3 ubiquitination of the non-functional 80S ribosome induces subunit dissociation by Slh1, leading to degradation of the non-functional 18S rRNA.

Pathophysiological roles of autophagy and aldo-keto reductases in development of doxorubicin resistance in gastrointestinal cancer cells.

Here, we show that incubation of three human gastrointestinal cancer cell lines (HCT15, LoVo and MKN45) with doxorubicin (DOX) provokes autophagy through facilitating production of reactive oxygen species (ROS). HCT15 cell treatment with DOX resulted in up-regulation of Beclin1, down-regulation of Bcl2, activation of AMPK and JNK, and Akt inactivation, all of which were restored by pretreating with an antioxidant N-acetyl-l-cysteine. These data suggest that all the autophagy-related alterations evoked by DOX result from the ROS production. In the DOX-resistant cancer cells, degree of autophagy elicited by DOX was milder than the parental cells, and DOX treatment hardly activated the ROS-dependent apoptotic signals [formation of 4-hydroxy-2-nonenal (HNE), cytochrome-c release into cytosol, and activation of JNK and caspase-3], inferring an inverse correlation between cellular antioxidant capacity and autophagy induction by DOX. Monitoring of expression levels of aldo-keto reductases (AKRs) in the parental and DOX-resistant cells revealed an up-regulation of AKR1B10 and/or AKR1C3 with acquiring the DOX resistance. Knockdown and inhibition of AKR1B10 or AKR1C3 in these cells enhanced DOX-elicited autophagy. Measurement of DOX-reductase activity and HNE-sensitivity assay also suggested that both AKR1B10 (via high HNE-reductase activity) and AKR1C3 (via low HNE-reductase and DOX-reductase activities) are involved in the development of DOX resistance. Combination of inhibitors of autophagy and the two AKRs overcame DOX resistance and cross-resistance of gastrointestinal cancer cells with resistance development to DOX or cis-diamminedichloroplatinum. Therefore, concomitant treatment with the inhibitors may be effective as an adjuvant therapy for elevating DOX sensitivity of gastrointestinal cancer cells.

Mutational analysis of the conserved carboxylates of anion channelrhodopsin-2 (ACR2) expressed in Escherichia coli and their roles in anion transport.

Anion channelrhodopsin-2 (ACR2), a light-gated channel recently identified from the cryptophyte alga Guillardia theta, exhibits anion channel activity with exclusive selectivity. In addition to its novel function, ACR2 has become a focus of interest as a powerful tool for optogenetics. Here we combined experimental and computational approaches to investigate the roles of conserved carboxylates on the anion transport activity of ACR2 in Escherichia coli membrane. First, we replaced six conserved carboxylates with a neutral residue (i.e. E9Q, E56Q, E64Q, E159Q, E219Q and D230N), and measured anion transport activity using E. coli expression system. E159Q and D230N exhibited significantly lower anion transport activity compared with wild-type ACR2 (1/12~1/3.4), which suggests that E159 and D230 play important roles in the anion transport. Second, to explain its molecular aspects, we constructed a homology model of ACR2 based on the crystal structure of a cation channelrhodopsin (ChR). The model structure showed a cavity formed by four transmembrane helices (TM1, TM2, TM3 and TM7) similar to ChRs, as a putative anion conducting pathway. Although E159 is not located in the putative pathway, the model structure showed hydrogen bonds between E159 and R129 with a water molecule. D230 is located in the pathway near the protonated Schiff base (PSB) of the chromophore retinal, which suggests that there is an interaction between D230 and the PSB. Thus, we demonstrated the functional importance and the hypothetical roles of two conserved carboxylates, E159 and D230, in the anion transport activity of ACR2 in E. coli membrane.

Oncocytic carcinoma of the salivary gland in a dog.

A 3-y-old male miniature Dachshund was presented with an ~0.8 cm diameter mass in the right mandibular region. Fourteen months later, the mass was 5 × 4 × 3 cm. Grossly, the mass was encapsulated and was homogeneously gray-white on cut surface. Microscopically, the mass was composed of large, round to polygonal tumor cells that were arranged in solid nests and cords separated by a fibrovascular stroma. Tumor cells had large, round, hypochromatic nuclei containing large prominent nucleoli and abundant eosinophilic cytoplasm containing dark blue granules visible with phosphotungstic acid-hematoxylin stain. Metastasis was observed in the mandibular lymph node. Immunohistochemically, tumor cells were positive for CK AE1/AE3, low-molecular-weight CK (CAM5.2), E-cadherin, mitochondria ATPase beta subunit, and S100, but were negative for vimentin, carcinoembryonic antigen, p63, CK14, CD10, and chromogranin A. Ultrastructurally, tumor cells contained numerous mitochondria. Therefore, the tumor was diagnosed as an oncocytic carcinoma of the mandibular gland.

The PI3K/mTOR dual inhibitor NVP-BEZ235 reduces the growth of ovarian clear cell carcinoma.

Patients with clear cell carcinoma of the ovary (OCCC) have poor survival due to resistance to standard chemotherapy. OCCC has frequent activating mutations of the PIK3CA gene. The present study was conducted to clarify the efficacy of the inhibition of the PI3K-AKT-mTOR pathway in OCCC. We used 8 OCCC cell lines and 5 ovarian serous adenocarcinoma (OSAC) cell lines. The mutation status of the PIK3CA and KRAS genes was examined by direct sequencing. The IC50 values of NVP-BEZ235 (BEZ235) and temsirolimus were determined by WST-8 assay. Protein expression levels of PI3K-AKT-mTOR pathway molecules were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Annexin V staining was used for detecting apoptosis. We also investigated the effects of BEZ235 on OCCC tumor growth in a nude mouse xenograft model. Four of the 8 OCCC cell lines showed a PIK3CA mutation while none of the 5 OSAC cell lines showed a mutation. The IC50 values of BEZ235 for the OCCC cell lines were lower than these values for the OSAC cell lines. The IC50 value of temsirolimus was higher than BEZ235 in the OCCC cell lines. The PIK3CA mutation was more frequently noted in OCCC than OSAC cells, but the sensitivity of these cell lines to BEZ235 or temsirolimus was not related to the mutation status. pHER3 and pAkt proteins were expressed more frequently in OCCC compared with OSAC. However, protein expression levels were distributed widely, and were not related to the sensitivity. Treatment with BEZ235 suppressed expression of pAkt, although treatment with temsirolimus did not. OCCC cells exhibited G1 phase arrest after treatment with BEZ235 and apoptosis with a higher concentration of the agent. BEZ235 significantly inhibited tumor growth in mice bearing OVISE and TU-OC-1 cell tumors. The present study indicated that the PI3K-AKT-mTOR pathway is a potential target for OCCC, and that BEZ235 warrants investigation as a therapeutic agent.

Establishment and characterization of a novel ovarian clear cell adenocarcinoma cell line, TU-OC-1, with a mutation in the PIK3CA gene.

A new cell line of human ovarian clear cell adenocarcinoma (CCC), TU-OC-1, was established and characterized. The cells showed a polygonal-shaped morphology and grew in monolayers without contact inhibition and were arranged like a jigsaw puzzle. The chromosome numbers ranged from 64 to 90. A low rate of proliferation was observed, similar to other CCC cell lines tested (OVTOKO, RMG-I, and OVAS), and the doubling time was 38.4 h. The respective IC50 values of cisplatin and paclitaxel were 12.2 µM and 58.3 nM. Mutational analysis revealed that TU-OC-1 cells harbored a PIK3CA mutation at codon 542 (E542K) in exon 9, which is a mutation hot spot on this gene. We observed that phosphorylated Akt protein was overexpressed in TU-OC-1 cells by western blot analysis. Heterotransplantation to nude mice produced tumors that reflected the original. This cell line may be useful to study the chemoresistant mechanisms of CCC and contribute to novel treatment strategies.

Establishment and characterization of a novel ovarian serous adenocarcinoma cell line, TU-OS-4, that overexpresses EGFR and HER2.

A new line of human ovarian serous adenocarcinoma cells, TU-OS-4, was established and characterized. The cells showed a short, spindle-shaped morphology and grew in monolayers without contact inhibition while forming an arrangement resembling a jigsaw puzzle. Chromosome numbers ranged from 55 to 73. The proliferation rate was lower than other serous adenocarcinoma cell lines tested (KF, SHIN-3, and SK-OV-3), and the doubling time was 53.3 h. Western blot analysis showed that TU-OS-4 cells overexpressed epidermal growth factor receptor, human epidermal growth factor receptor (HER) 2, and phosphorylated HER2 protein. The IC(50) values to cisplatin, paclitaxel, and lapatinib were 25.8 µM, 686 nM, and 183 nM, respectively. Heterotransplantation in nude mice reflected the original tumor of the cells. These results suggested that this cell line would be useful to study chemoresistant mechanisms and contribute to establishing novel treatment strategies for patients with ovarian cancer.