Analysis of the susceptibility of refractory hepatitis C virus resistant to nonstructural 5A inhibitors.

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.

Isolation and Structure Determination of New Pyrones from Dictyostelium spp. Cellular Slime Molds Coincubated with Pseudomonas spp.

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.

Glycoprotein non-metastatic melanoma protein B expression correlates with the prognosis of acute liver injury/failure.

Background: Glycoprotein non-metastatic melanoma protein B (GPNMB) is expressed in macrophages during recovery from acute liver injury (ALI) in carbon tetrachloride (CCl4)-induced liver injury model mice. In this retrospective study, we assessed whether GPNMB levels in the serum and injured liver correlate with liver injury severity and prognosis in patients with ALI or acute liver failure (ALF). Methods: The study involved 56 patients with ALI or ALF who visited the Kagoshima University Hospital. Serum GPNMB level was measured over time, and the localization, proportion, origin, and phenotype of GPNMB-expressing cells in the injured liver were assessed. Finally, the phenotypes of human monocyte-derived macrophages and peripheral blood mononuclear cells (PBMCs) of patients with ALI and ALF were analyzed. Results: Peak GPNMB levels were significantly higher in patients with ALF and hepatic encephalopathy (HE), as well as in those who underwent liver transplantation or died, than in others. The peak GPNMB level correlated with prothrombin activity, prothrombin time-international normalized ratio, Model for End-stage Liver Disease score, and serum hepatocyte growth factor level. GPNMB was expressed in CD68-positive macrophages, and its level increased with the severity of liver injury. The macrophages showed the same polarization as M2c macrophages induced with interleukin-10 from human monocytes. Moreover, PBMCs from patients with ALF exhibited an immunosuppressive phenotype. Conclusion: We found that GPNMB levels in the serum and injured liver, which increased in patients with ALF, especially in those with HE, correlated with the severity of liver injury and prognosis of ALI and ALF. Moreover, GPNMB-positive macrophages exhibited the M2c phenotype. Our results indicate that persistently high GPNMB levels may be a prognostic marker in patients with ALI and ALF.

Factors affecting patient safety culture in a university hospital under the universal health insurance system: A cross-sectional study from Japan.

We conducted a cross-sectional study of patient safety culture aimed at examining the factors that influence patient safety culture in university hospitals under a universal health insurance system. The Hospital Survey on Patient Safety Culture developed by the Agency for Healthcare Research and Quality was used. The survey was distributed to 1066 hospital employees, and 864 responded. The confirmatory factor analysis showed a good fit of the results to the 12-composites model. The highest positive response rates were for "(1) Teamwork within units" (81%) and "(2) Supervisor/manager expectations and actions promoting patient safety" (80%), and the lowest was for "(10) Staffing" (36%). Hayashi's quantification theory type 2 revealed that working hours per week had the greatest negative impact on patient safety culture. Under a universal health insurance system, workload and human resources might have a significant impact on the patient safety culture.

HGF-MET Signaling Shifts M1 Macrophages Toward an M2-Like Phenotype Through PI3K-Mediated Induction of Arginase-1 Expression.

Backgrounds and Aims: Hepatocyte Growth Factor (HGF)-MET signaling is known to promote biological functions such as cell survival, cell motility, and cell proliferation. However, it is unknown if HGF-MET alters the macrophage phenotype. In this study, we aimed to study the effects of HGF-MET signaling on the M1 macrophage phenotype. Methods and Materials: Bone marrow-derived macrophages (BMDMs) isolated from mice were either polarized to an M1 phenotype by IFN-γ and LPS treatment or to an M2 phenotype by IL-4 treatment. Changes in M1 or M2 markers induced by HGF-MET signaling were evaluated. Mechanisms responsible for alternations in the macrophage phenotype and intracellular metabolism were analyzed. Results: c-Met was expressed especially in M1 macrophages polarized by treatment with IFN-γ and LPS. In M1 macrophages, HGF-MET signaling induced the expression of Arg-1 mRNA and secretion of IL-10 and TGF-ß1 and downregulated the mRNA expression of iNOS, TNF-α, and IL-6. In addition, activation of the PI3K pathway and inactivation of NFκB were also observed in M1 macrophages treated with HGF. The increased Arg-1 expression and IL-10 secretion were abrogated by PI3K inhibition, whereas, no changes were observed in TNF-α and IL-6 expression. The inactivation of NFκB was found to be independent of the PI3K pathway. HGF-MET signaling shifted the M1 macrophages to an M2-like phenotype, mainly through PI3K-mediated induction of Arg-1 expression. Finally, HGF-MET signaling also shifted the M1 macrophage intracellular metabolism toward an M2 phenotype, especially with respect to fatty acid metabolism. Conclusion: Our results suggested that HGF treatment not only promotes regeneration in epithelial cells, but also leads to tissue repair by altering M1 macrophages to an M2-like phenotype.

Two New Terpenes Isolated from Dictyostelium Cellular Slime Molds.

We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.

Role of OAT4 in Uptake of Estriol Precursor 16α-Hydroxydehydroepiandrosterone Sulfate Into Human Placental Syncytiotrophoblasts From Fetus.

Estriol biosynthesis in human placenta requires the uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT)-4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [(3)H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hours but was inhibited in the presence of 50 µM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.

Autopsy report on central pontine myelinolysis triggered by vomiting associated with digoxin intoxication.

An 87-year-old male, prescribed digoxin and furosemide for congestive heart failure and Alzheimer disease, had dehydration and anemia due to poor food intake and hemorrhagic cystitis. Repeated vomiting due to an upper respiratory infection caused disturbance of consciousness and hypotension. The patient was admitted to hospital and diagnosed with digoxin intoxication and hypernatremia. The serum sodium (Na(+)) level was corrected, but the patient died 4 days after admission following uncontrollable seizure. A histologic examination after an autopsy revealed characteristic findings of central pontine myelinolysis (CPM). This is the first autopsy report on CPM triggered by vomiting in association with digoxin administration.

Three-dimensional reconstructive analysis of intracytoplasmic lumina found in ependymomas.

Three-dimensional reconstructive analyses revealed that the intracytoplasmic lumina found in ependymomas were actually formed by subsidence of an extracellular membrane, resembling a volcano. This finding was compatible with cytologic and electron microscopic findings. In addition, there were many tiny thorns resembling a holly leaf on the extracellular membrane, such that cilia and microvilli on the cellular membrane discontinued cell-to-cell tight junctions.