Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.     

Genomic characterization of closely related species in the Rumoiensis clade infers ecogenomic signatures to non-marine environments

Members of the family Vibrionaceae are generally found in marine and brackish environments, playing important roles in nutrient cycling. The Rumoiensis clade is an unconventional group in the genus Vibrio, currently comprising six species from different origins including two species isolated from non-marine environments. In this study, we performed comparative genome analysis of all six species in the clade using their complete genome sequences. We found that two non-marine species, Vibrio casei and Vibrio gangliei, lacked the genes responsible for algal polysaccharide degradation, while a number of glycoside hydrolase genes were enriched in these two species. Expansion of insertion sequences was observed in V. casei and Vibrio rumoiensis, which suggests ongoing genomic changes associated with niche adaptations. The genes responsible for the metabolism of glucosylglycerate, a compound known to play a role as compatible solutes under nitrogen limitation, were conserved across the clade. These characteristics, along with genes encoding species-specific functions, may reflect the habit expansion which has led to the current distribution of Rumoiensis clade species. Genome analysis of all species in a single clade give us valuable insights into the genomic background of the Rumoiensis clade species and emphasize the genomic diversity and versatility of Vibrionaceae.     

Mass balance analysis of contaminated heparin product

A quantitative analysis of a recalled contaminated lot of heparin sodium injection U.S. Pharmacopeia (USP) was undertaken in response to the controversy regarding the exact nature of the contaminant involved in the heparin (HP) crisis. A mass balance analysis of the formulated drug product was performed. After freeze-drying, a 1-ml vial for injection afforded 54.8 ± 0.3 mg of dry solids. The excipients, sodium chloride and residual benzyl alcohol, accounted for 11.4 ± 0.5 and 0.9 ± 0.5 mg, respectively. Active pharmaceutical ingredient (API) represented 41.5 ± 1.0 mg, corresponding to 75.7 wt% of dry mass. Exhaustive treatment of API with specific enzymes, heparin lyases, and/or chondroitin lyases was used to close mass balance. HP represented 30.5 ± 0.5 mg, corresponding to 73.5 wt% of the API. Dermatan sulfate (DS) impurity represented 1.7 ± 0.3 mg, corresponding to 4.1 wt% of API. Contaminant, representing 9.3 ± 0.1 mg corresponding to 22.4 wt% of API, was found in the contaminated formulated drug product. The recovery of contaminant was close to quantitative (95.6–100 wt%). A single contaminant was unambiguously identified as oversulfated chondroitin sulfate (OSCS).     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Molecular identification of GHS-R and GPR38 in Suncus murinus

We previously identified ghrelin and motilin genes in Suncus murinus (suncus), and also revealed that motilin induces phase III-like strong contractions in the suncus stomach in vivo, as observed in humans and dogs. Moreover, repeated migrating motor complexes were found in the gastrointestinal tract of suncus at regular 120-min intervals. We therefore proposed suncus as a small laboratory animal model for the study of gastrointestinal motility. In the present study, we identified growth hormone secretagogue receptor (GHS-R) and motilin receptor (GPR38) genes in the suncus. We also examined their tissue distribution throughout the body. The amino acids of suncus GHS-R and GPR38 showed high homology with those of other mammals and shared 42% amino acid identity. RT-PCR showed that both the receptors were expressed in the hypothalamus, medulla oblongata, pituitary gland and the nodose ganglion in the central nervous system. In addition, GHS-R mRNA expressions were detected throughout the stomach and intestine, whereas GPR38 was expressed in the gastric muscle layer, lower intestine, lungs, heart, and pituitary gland. These results suggest that ghrelin and motilin affect gut motility and energy metabolism via specific receptors expressed in the gastrointestinal tract and/or in the central nervous system of suncus.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

“Green odor” inhalation by stressed rat dams reduces behavioral and neuroendocrine signs of prenatal stress in the offspring

Chronic maternal stress during pregnancy results in the “prenatally stressed” offspring displaying behavioral and neuroendocrine alterations that persist into adulthood. We investigated how inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) by stressed dams might alter certain indices of prenatal stress in their offspring. These indices were depression-like behavior (increased immobility time in the forced-swim test) and acute restraint stress-induced changes in hypothalamo-pituitary-adrenocortical (HPA) axis activity [plasma corticosterone (CORT) and ACTH levels and the number of Fos-immunoreactive cells in the hypothalamic paraventricular nucleus (an index of neuronal activity)]. Pregnant rats were exposed to restraint stress for 60 min/day for 10 days (gestational days 10-19). The prenatally stressed offspring exhibited significant increases in depression-like behavior and in restraint stress-induced ACTH, CORT, and Fos responses, unless their dam had been exposed to green odor. The behavioral effect of the odor was also seen in offspring that were fostered by unstressed dams. The results obtained in the dams themselves were as follows. In vehicle-exposed stressed dams, but not in green odor-exposed ones, total body and adrenal weights were significantly decreased or increased, respectively. Depression-like behavior was not observed in the vehicle-exposed stressed dams themselves. Green odor inhalation prevented the impairment of maternal behavior induced by restraint stress. Thus, exposure of dams to stress may affect both the fetal brain and fetal HPA axis, and also maternal behavior, leading to altered behavioral and neuroendocrine responses in the offspring. Such effects may be prevented by the stressed dams inhaling green odor.     

Identification of a novel actin-related protein in Tetrahymena cilia

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In eukaryotes, its gene belongs to a highly conserved gene family, while the protein is also a member of an actin superfamily comprising various kinds of actin-related proteins (Arps). A ciliate, Tetrahymena, has a unique conventional actin. Data from the TIGR Tetrahymena genome project and our own research suggest the existence of 12 actin-like sequences: four conventional actins, two of Arp4, one each of Arp1, Arp2, Arp3, Arp5, and Arp6, and a novel actin-related protein, tArp. We cloned the entire cDNA sequences of Tetrahymena Arp2 (tArp2), Tetrahymena Arp3 (tArp3), and tArp for the work described herein. In phylogenetic analyses, tArp was not included in any Arp subfamily. Unlike other known Arps, tArp localizes in cilia, and its expression was upregulated after deciliation. To see the precise localization of tArp, cilia were fractionated and analyzed using specific antibodies. tArp was observed preferentially in the "outer-doublet" fraction, while actin was found in the "crude-dynein" fraction. In immunoelectron microscopy, most of the gold particles were found either on the outer-doublet or central-pair microtubules. These results suggest that tArp is a ciliary component and that it has a unique function in the formation and maintenance of cilia.     

Absorption of Radionuclides from the Fukushima Nuclear Accident by a Novel Algal Strain

Large quantities of radionuclides have leaked from the Fukushima Daiichi Nuclear Power Plant into the surrounding environment. Effective prevention of health hazards resulting from radiation exposure will require the development of efficient and economical methods for decontaminating radioactive wastewater and aquatic ecosystems. Here we describe the accumulation of water-soluble radionuclides released by nuclear reactors by a novel strain of alga. The newly discovered green microalgae, Parachlorella sp. binos (Binos) has a thick alginate-containing extracellular matrix and abundant chloroplasts. When this strain was cultured with radioiodine, a light-dependent uptake of radioiodine was observed. In dark conditions, radioiodine uptake was induced by addition of hydrogen superoxide. High-resolution secondary ion mass spectrometry (SIMS) showed a localization of accumulated iodine in the cytosol. This alga also exhibited highly efficient incorporation of the radioactive isotopes strontium and cesium in a light-independent manner. SIMS analysis showed that strontium was distributed in the extracellular matrix of Binos. Finally we also showed the ability of this strain to accumulate radioactive nuclides from water and soil samples collected from a heavily contaminated area in Fukushima. Our results demonstrate that Binos could be applied to the decontamination of iodine, strontium and cesium radioisotopes, which are most commonly encountered after nuclear reactor accidents.