Development of a fluorescence reporter system to quantify transcriptional activity of endogenous p53 in living cells.

The tumor suppressor p53 (also known as TP53) plays a central role in cellular stress responses by regulating transcription of multiple target genes. The temporal dynamics of p53 are thought to be important for its function; these encode input information and are decoded to induce distinct cellular phenotypes. However, it remains unclear to what extent the temporal dynamics of p53 reflect the activity of p53-induced gene expression. In this study, we report a multiplexed reporter system that allows us to visualize the transcriptional activity of p53 at the single-cell level. Our reporter system features simple and sensitive observation of the transcriptional activity of endogenous p53 to the response elements of various target genes. Using this system, we show that the transcriptional activation of p53 exhibits strong cell-to-cell heterogeneity. The transcriptional activation of p53 after etoposide treatment is highly dependent on the cell cycle but this is not seen after UV exposure. Finally, we show that our reporter system allows simultaneous visualization of the transcriptional activity of p53 and cell cycle. Our reporter system can thus be a useful tool for studying biological processes involving the p53 signaling pathway.

Clinical significance of serum cytokine profiles for differentiating between Kawasaki disease and its mimickers.

OBJECTIVES: To investigate the clinical significance of serum cytokine profiles for differentiating between Kawasaki disease (KD) and its mimickers. METHODS: Patients with KD, including complete KD, KD shock syndrome (KDSS), and KD with macrophage activation syndrome (KD-MAS), and its mimickers, including multisystem inflammatory syndrome in children, toxic shock syndrome, and Yersinia pseudotuberculosis infection, were enrolled. Serum levels of interleukin (IL)-6, soluble tumor necrosis factor receptor type II (sTNF-RII), IL-10, IL-18, and chemokine (C-X-C motif) ligand 9 (CXCL9) were measured using enzyme-linked immunosorbent assay and compared them with clinical manifestations. RESULTS: Serum IL-6, sTNF-RII, and IL-10 levels were significantly elevated in patients with KDSS. Serum IL-18 levels were substantially elevated in patients with KD-MAS. Patients with KD-MAS and KD mimickers had significantly elevated serum CXCL9 levels compared with those with complete KD. Area under the receiver operating characteristic curve analysis showed that serum IL-6 was the most useful for differentiating KDSS from the others, IL-18 and CXCL9 for KD-MAS from complete KD, and CXCL9 for KD mimickers from complete KD and KD-MAS. CONCLUSION: Serum cytokine profiles may be useful for differentiating between KD and its mimickers.

Clinical Usefulness of T-Cell Receptor Vß Repertoire Analysis for Differentiating Multisystem Inflammatory Syndrome in Japanese Children From Toxic Shock Syndrome and Kawasaki Disease.

The specific expansion of T-cell receptor ß chain variable region (TCR-Vß21.3 + ) CD4 + and CD8 + T cells was observed in Japanese patients with multisystem inflammatory syndrome in children. In contrast, these findings were not observed in patients with toxic shock syndrome and Kawasaki disease. T-cell receptor ß chain variable region repertoire analysis to detect specific expansion of Vß21.3 + T cells might be useful for differentiating multisystem inflammatory syndrome in children from toxic shock syndrome and Kawasaki disease.

Sensorineural Hearing Loss due to Acute Carbon Monoxide Poisoning.

Carbon monoxide (CO) can cause "irreversible" severe-to-profound sensorineural hearing loss. However, there are few reports of detailed hearing test results. Here, we report a case of acute sensorineural hearing loss caused by acute CO poisoning with partial hearing recovery, evaluated by a detailed hearing examination. A 25-year-old woman was brought to the emergency department for attempted suicide. On admission, her consciousness was impaired, and she was treated for severe CO poisoning, including using hyperbaric-oxygen therapy. After regaining consciousness, symptoms of hearing loss and tinnitus were discovered, and a detailed audiological examination revealed bilateral hearing loss, suggesting cochlear damage. Steroids were systemically administered, and her hearing impairment was partially resolved. Sensorineural hearing loss caused by acute CO poisoning includes cochlear pathology and may be partially treatable. The early evaluation of hearing in patients with severe CO poisoning is advisable for early treatment.

Characteristics of aspiration pneumonia patients in acute care hospitals: A multicenter, retrospective survey in Northern Japan.

BACKGROUND: Pneumonia is a common cause of illness and death of the elderly in Japan. Its prevalence is escalating globally with the aging of population. To describe the latest trends in pneumonia hospitalizations, especially aspiration pneumonia (AP) cases, we assessed the clinical records of pneumonia patients admitted to core acute care hospitals in Miyagi prefecture, Japan. METHODS: A retrospective multi-institutional joint research was conducted for hospitalized pneumonia patients aged ≥20 years from January 2019 to December 2019. Clinical data of patients were collected from the medical records of eight acute care hospitals. RESULTS: Out of the 1,800 patients included in this study, 79% of the hospitalized pneumonia patients were aged above 70 years. The most common age group was in the 80s. The ratio of AP to total pneumonia cases increased with age, and 692 out of 1,800 patients had AP. In univariate analysis, these patients had significantly older ages, lower body mass index (BMI), a lower ratio of normal diet intake and homestay before hospitalization, along with more AP recurrences and comorbidities. During hospitalization, AP patients had extended fasting periods, more swallowing assessments and interventions, longer hospitalization, and higher in-hospital mortality rate than non-AP patients. A total of 7% and 2% AP patients underwent video endoscopy and video fluorography respectively. In multivariate analysis, lower BMI, lower C-reactive protein, a lower ratio of homestay before hospitalization, a higher complication rate of cerebrovascular disease, dementia, and neuromuscular disease were noted as a characteristic of AP patients. Swallowing interventions were performed for 51% of the AP patients who had been hospitalized for more than two weeks. In univariate analysis, swallowing intervention improved in-hospital mortality. Lower AP recurrence before hospitalization and a lower ratio of homestay before hospitalization were indicated as characteristics of AP patients of the swallowing intervention group from multivariate analysis. Change in dietary pattern from normal to modified diet was observed more frequently in the swallowing intervention group. CONCLUSION: AP accounts for 38.4% of all pneumonia cases in acute care hospitals in Northern Japan. The use of swallowing evaluations and interventions, which may reduce the risk of dysphagia and may associate with lowering mortality in AP patients, is still not widespread.

New frontiers in gut nutrient sensor research: prophylactic effect of glutamine against Helicobacter pylori-induced gastric diseases in Mongolian gerbils.

Ammonia is one of the important toxins produced by Helicobacter pylori (H. pylori), the major cause of peptic ulcer diseases. We examined whether glutamine or marzulene (a gastroprotective drug containing 1% sodium azulene and 99% glutamine) protects the gastric mucosa against H. pylori in vivo and investigated the mechanism underlying glutamine-induced mucosal protection against ammonia in gastric epithelial cells in vitro. Mongolian gerbils were fed for 3 months with a diet containing glutamine (2%-20%) or marzulene (20%) starting from 2 weeks or 2 years after H. pylori infection. Then, gastric mucosal changes were evaluated both macro- and microscopically. Cultured gastric epithelial cells were incubated in the presence of ammonia, with or without glutamine; and cell viability, ammonia accumulation, and chemokine production were determined. Gerbils exhibited edema, congestion, and erosion after 3-month infection; and after 2-year infection, they showed cancer-like changes in the gastric mucosa. Glutamine and marzulene significantly suppressed these pathological changes caused in the gastric mucosa by H. pylori infection. Ammonia was accumulated in the cells, resulting in an increase in chemokine production and a decrease in cell viability. These pathological responses were prevented by glutamine. In addition, glutamine decreased chemokine production and cell death through inhibition of cellular accumulation of ammonia, resulting in the prevention of H. pylori-induced gastric diseases in vivo. These results suggest that glutamine/marzulene would be useful for prophylactic treatment of H. pylori-induced gastric diseases in patients.

Collimated Microbeam Reveals that the Proportion of Non-Damaged Cells in Irradiated Blastoderm Determines the Success of Development in Medaka (Oryzias latipes) Embryos.

It has been widely accepted that prenatal exposure to ionizing radiation (IR) can affect embryonic and fetal development in mammals, depending on dose and gestational age of the exposure, however, the precise machinery underlying the IR-induced disturbance of embryonic development is still remained elusive. In this study, we examined the effects of gamma-ray irradiation on blastula embryos of medaka and found transient delay of brain development even when they hatched normally with low dose irradiation (2 and 5 Gy). In contrast, irradiation of higher dose of gamma-rays (10 Gy) killed the embryos with malformations before hatching. We then conducted targeted irradiation of blastoderm with a collimated carbon-ion microbeam. When a part (about 4, 10 and 25%) of blastoderm cells were injured by lethal dose (50 Gy) of carbon-ion microbeam irradiation, loss of about 10% or less of blastoderm cells induced only the transient delay of brain development and the embryos hatched normally, whereas embryos with about 25% of their blastoderm cells were irradiated stopped development at neurula stage and died. These findings strongly suggest that the developmental disturbance in the IR irradiated embryos is determined by the proportion of severely injured cells in the blastoderm.

The role of hydrophobic amino acids of K-segments in the cryoprotection of lactate dehydrogenase by dehydrins.

Dehydrins, which are group 2 late embryogenesis abundant (LEA) proteins, accumulate in plants during the development of the embryo and exposure to abiotic stresses including low temperature. Dehydrins exhibit cryoprotection of freezing-sensitive enzymes, e.g. lactate dehydrogenase (LDH). Although it has been reported that K-segments conserved in dehydrins are related to their cryoprotection activity, it has not been determined which sequence features of the K-segments contribute to the cryoprotection. A cryoprotection assay using LDH indicated that 13 K-segments including 12 K-segments found in Arabidopsis dehydrins and a typical K-segment (TypK, EKKGIMEKIKEKLPG) derived from the K-segments of many plants showed similar cryoprotective activities. Mutation of the TypK sequence demonstrated that hydrophobic amino acids were clearly involved in preventing the cryoinactivation, cryoaggregation, and cryodenaturation of LDH. We propose that the cryoprotective activities of dehydrins may be made possible by the hydrophobic residues of the K-segments.

Comprehensive characterization of temperature- and pressure-induced bilayer phase transitions for saturated phosphatidylcholines containing longer chain homologs.

Complete elucidation of the phase behavior of phospholipid bilayers requires information on the subtransition from the lamellar crystal (Lc) phase to the gel phase. However, for bilayers of saturated diacylphosphatidylcholines (CnPCs), especially longer chain homologs, equilibration in the Lc phase is known to be very slow. In this study, bilayer phase transitions of three CnPCs with longer acyl chains, C19PC, C20PC and C21PC, were observed by differential scanning calorimetry under atmospheric pressure and by light-transmittance measurements under high pressure. Using lipid samples treated by thermal annealing enabled the observation of the sub-, pre- and main transitions of the C19PC and C20PC bilayers under atmospheric pressure. Only the pre- and main transitions could be observed for the C21PC bilayer due to very slow kinetics of the Lc phase formation for lipids with long acyl chains. The temperature and pressure phase diagrams constructed and phase-transitions quantities (enthalpy, entropy and volume changes) evaluated for these bilayers were compared with one another and with those of bilayers of the CnPC homologs examined in previous studies. These results allowed us (1) to clarify the temperature- and pressure-dependent phase sequence and phase stability of the CnPC (n=12-22) bilayers as a function of the hydrophobicity of the molecules, (2) to prove the presence of a shorter and a longer limit (n=13 and 21) in the acyl chain length for the pressure-induced bilayer interdigitation and (3) to reveal the chain-length dependence of the thermodynamic quantities of the subtransitions including the volume change.