The clinical usage of liposomal amphotericin B in patients receiving renal replacement therapy in Japan: a nationwide observational study.

BACKGROUND: Liposomal amphotericin B (L-AMB), a broad-spectrum antifungicidal drug, is often used to treat fungal infections. However, clinical evidence of its use in patients with renal dysfunction, especially those receiving renal replacement therapy (RRT), is limited. Therefore, we evaluated the usage and occurrence of adverse reactions during L-AMB therapy in patients undergoing RRT. METHODS: Using claims data and laboratory data, we retrospectively evaluated patients who were administered L-AMB. The presence of comorbidities, mortality rate, treatment with L-AMB and other anti-infective agents, and the incidence of adverse reactions were compared between patients receiving RRT, including continuous renal replacement therapy (CRRT) and maintenance hemodialysis (HD), and those that did not receive RRT. RESULTS: In total, 900 cases met the eligibility criteria: 24, 19, and 842 cases in the maintenance HD, CRRT, and non-RRT groups, respectively. Of the patients administered L-AMB, mortality at discharge was higher for those undergoing either CRRT (15/19; 79%) or maintenance HD (16/24; 67%) than for those not receiving RRT (353/842; 42%). After propensity score matching, the average daily and cumulative dose, treatment duration, and dosing interval for L-AMB were not significantly different between patients receiving and not receiving RRT. L-AMB was used as the first-line antifungal agent for patients undergoing CRRT in most cases (12/19; 63%). Although the number of subjects was limited, the incidence of adverse events did not markedly differ among the groups. CONCLUSION: L-AMB may be used for patients undergoing maintenance HD or CRRT without any dosing, duration, or interval adjustments.

Efficacy of early administration of liposomal amphotericin B in patients with septic shock: A nationwide observational study.

INTRODUCTION: Liposomal amphotericin B (L-AMB), a broad spectrum anti-fungicidal drug, is often administered to treat invasive fungal infections (IFIs). However, the most suitable time to initiate treatment in septic shock patients with IFI is unknown. METHODS: Patients with septic shock treated with L-AMB were identified from the Japanese Diagnosis Procedure Combination national database and were stratified according to L-AMB treatment initiation either at septic shock onset (early L-AMB group) or after the onset (delayed L-AMB group) to determine their survival rates following septic shock onset and the shock cessation period. RESULTS: We identified 141 patients administered L-AMB on the day of or after septic shock onset: 60 patients received early treatment, whereas 81 patients received delayed treatment. Survival rates after septic shock onset were higher in the early L-AMB group than in the delayed L-AMB group (4 weeks: 68.4% vs 57.9%, P = 0.197; 6 weeks: 62.2% vs 44.5%, P = 0.061; 12 weeks: 43.4% vs 35.0%, P = 0.168, respectively). The septic shock cessation period was shorter in the early L-AMB group than in the delayed L-AMB group (7.0 ± 7.0 days vs 16.5 ± 15.4 days, P < 0.001), with a significant difference confirmed after adjusting for confounding factors with propensity score matching (7.1 ± 7.2 days vs 16.7 ± 14.0 days, P = 0.001). CONCLUSION: Early L-AMB administration at septic shock onset may be associated with early shock cessation.

Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient.

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.

BACKGROUND: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination. METHODS: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection. RESULTS: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites. CONCLUSIONS: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.

Seismic constraints on rotation of Sun-like star and mass of exoplanet.

Rotation is thought to drive cyclic magnetic activity in the Sun and Sun-like stars. Stellar dynamos, however, are poorly understood owing to the scarcity of observations of rotation and magnetic fields in stars. Here, inferences are drawn on the internal rotation of a distant Sun-like star by studying its global modes of oscillation. We report asteroseismic constraints imposed on the rotation rate and the inclination of the spin axis of the Sun-like star HD 52265, a principal target observed by the CoRoT satellite that is known to host a planetary companion. These seismic inferences are remarkably consistent with an independent spectroscopic observation (rotational line broadening) and with the observed rotation period of star spots. Furthermore, asteroseismology constrains the mass of exoplanet HD 52265b. Under the standard assumption that the stellar spin axis and the axis of the planetary orbit coincide, the minimum spectroscopic mass of the planet can be converted into a true mass of 1.85(-0.42)(+0.52)M(Jupiter), which implies that it is a planet, not a brown dwarf.

Preparation, Structural Determination, and Characterization of Electronic Properties of Bis-silylated and Bis-germylated Lu3 N@Ih -C80.

Bis-silylated and bis-germylated derivatives of Lu3 N@Ih -C80 (3, 4, 5) were successfully synthesized by the photochemical addition of disiliranes 1 a, 1 b or digermirane 2, and fully characterized by spectroscopic, electrochemical, and theoretical studies. Interestingly, digermirane 2 reacts more efficiently than disiliranes 1 a and 1 b because of its good electron-donor properties and lower steric hindrance around the Ge-Ge bond. The 1,4-adduct structures of 3, 4, 5 were unequivocally established by single-crystal X-ray crystallographic analyses. The electrochemical and theoretical studies reveal that the energy gaps between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the 1,4-adducts are remarkably smaller than those of Lu3 N@Ih -C80 , because the electron-donating groups effectively raise the HOMO levels. It is also observed that germyl groups are slightly more electron-donating than the silyl groups on the basis of the redox properties and the HOMO-LUMO energies of 4 and 5. Bis-silylation and bis-germylation are effective and versatile methods for tuning the electronic characteristics of endohedral metallofullerenes.

Association between Tai Chi Yuttari Exercise and Longevity and Prevention of Long-Term Care Need: Survival Analysis in Kitakata City, Japan.

This study examined whether participation in Tai Chi Yuttari exercise is associated with a delay in the death and new certification for long-term care need of older adults. Individuals who participated in Tai Chi Yuttari exercise classes in 2011-2015 (participation group) were compared with individuals from the Basic Resident Register of Kitakata City (non-participation group). Death and new certification for long-term care need were selected to evaluate the effectiveness of participation in Tai Chi Yuttari exercise classes. The periods from the start date of the observation to each person's date of occurrence of events were calculated. The Kaplan-Meier method and log-rank test were used to compare survival curves between the groups. A total of 105 and 202 individuals in the participation and non-participation groups, respectively, were observed. Survival duration (χ2 = 8.782, p = 0.003) and the period before receiving certification for long-term care (χ2 = 5.354, p = 0.021) were longer in the participation group than in the non-participation group. In the stratified analysis by sex, survival duration was longer in the participation group in men only (χ2 = 7.875, p = 0.005). Participation in Tai Chi Yuttari exercise might be effective in delaying death, especially in men, and new certification for long-term care.

Synthesis of silylene-bridged endohedral metallofullerene Lu3N@I(h)-C80.

Functionalization of endohedral metallofullerenes has been shown to differ depending on photochemical or thermal pathways. We report that Lu(3)N@I(h)-C(80) reacts with thermally generated bis(2,6-diethylphenyl)silylene with high selectivity and forms monosilylated derivative 1b. Unexpectedly, 1b undergoes photochemical conversion to afford isomer 1a under ambient light. These adducts were characterized using NMR, visible-near-IR spectroscopy, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Single-crystal X-ray structure determination of 1a reveals a rare example of an open 1,2-adduct at the [5,6]-ring junction of the I(h)-C(80) cage. The electrochemical study reveals that the redox potentials of 1a and 1b are shifted cathodically compared to those of pristine Lu(3)N@I(h)-C(80) and that monosilylation is effective to fine-tune the electronic properties of endohedral metallofullerenes as well as empty fullerenes. Density functional theory calculations were also performed, which provide a theoretical basis for the structures and the behavior of the encapsulated Lu(3)N cluster.

Atypical metastatic carcinoid of the uterine cervix and review of the literature.

Atypical carcinoid tumors of the uterine cervix represent rare neuroendocrine tumors and are highly aggressive, showing early lymphatic invasion and hematogenous distant metastases. Because of the small number of cases, there are currently no recommendations regarding treatment, and little is known about the response to chemotherapeutic agents. A 39-year-old woman was diagnosed with a primary atypical carcinoid of the uterine cervix with numerous metastases to the liver. After radical hysterectomy, she underwent hepatic arterial chemoembolization with streptozotocin and 5-fluorouracil. Complete response was achieved in numerous liver metastases. At the 2-year follow up of chemotherapy, the patient remains alive. Treatment for atypical carcinoid tumors remains elusive, however hepatic arterial chemoembolization with streptozotocin and 5-fluorouracil was effective in the present primary atypical carcinoid with liver metastases. A review of the previous reports is also presented.

Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

Correlation between reduced susceptibility to disinfectants and multidrug resistance among clinical isolates of Acinetobacter species.

BACKGROUND: The aim of this study was to investigate the susceptibility profiles to disinfectants and antimicrobial agents of 283 non-repetitive Acinetobacter clinical isolates obtained in 97 Japanese hospitals in March 2002. METHODS: Susceptibility profiles of the above isolates to four disinfectants, six antimicrobial agents and two dyes were investigated. MICs were measured by the agar dilution method recommended by the CLSI (formerly NCCLS). MBC measurements and time-kill assays were performed using a slightly modified quantitative suspension test based on the European Standard EN 1040. RESULTS: No evident resistance to disinfectants was seen among the 283 strains of Acinetobacter spp. isolated in 2002, but the MIC(90)s of chlorhexidine gluconate, benzalkonium chloride and alkyldiaminoethylglycine hydrochloride were 50, 50 and 400 mg/L, respectively. Interestingly, the MICs of alkyldiaminoethylglycine hydrochloride and benzethonium chloride for four and three clinical isolates, respectively, reached 800 mg/L (approximately half the in-use concentration). The MBCs for the 28 disinfectant reduced susceptibility (DRS) isolates, for which the MICs of at least one of the four disinfectants tested were higher than the MIC(90), were comparable to those for susceptible isolates, in general; however, significant differences (P < 0.01) were observed between disinfectant-susceptible and DRS isolates in the time-kill assays of chlorhexidine gluconate, benzalkonium chloride and benzethonium chloride. Furthermore, DRS isolates tended to demonstrate multiresistance profiles to ceftazidime, ciprofloxacin and amikacin (P < 0.05). CONCLUSIONS: Since several Acinetobacter clinical isolates have developed augmented resistance to multiple antimicrobials and disinfectants, it is worth checking the susceptibility to disinfectants if multidrug-resistant Acinetobacter spp. are recurrently isolated clinically.

Matrix metalloproteinase-2 inhibitors from Clinopodium chinense var. parviflorum.

From the aerial parts of Clinopodium chinense var. parviflorum, nine new phenylpropanoids, clinopodic acids A-I (2-10), were isolated together with a known phenylpropanoid, rosmarinic acid (1). The structures of these new compounds were elucidated on the basis of spectroscopic analysis. Clinopodic acid C (4) showed MMP-2 inhibitory activity (IC(50) 3.26 microM).

Reduction of disinfectant bactericidal activities in clinically isolated Acinetobacter species in the presence of organic material.

OBJECTIVES: In clinical Acinetobacter species, the reduction effects of organic material on bactericidal activities of four major disinfectants were investigated: chlorhexidine gluconate (CHX), benzethonium chloride (BZT), benzalkonium chloride (BZK) and alkyl diaminoethyl glycine hydrochloride (ADH). METHODS: The bactericidal activities of the four disinfectants against 283 strains of Acinetobacter species recovered from 97 Japanese hospitals in March 2002 were investigated by four different tests: MIC measurements, MBC measurements, time-killing assays and adaptation assays. Moreover, disinfectant efficacy was examined in the presence of BSA in two tests: MBC measurements and time-killing assays. RESULTS: No clinical isolates were able to withstand the in-use concentrations of the four disinfectants, although the MIC90 of ADH reached 100 mg/L. Strains for which MICs of at least two disinfectants were higher than MIC90 measured by the broth microdilution method were defined as isolates with 'disinfectant reduced susceptibility (DRS)'. In the presence of 3.0% BSA, the MBCs of BZK, BZT and ADH for DRS isolates rose to 512 and 1024 mg/L, which were about half their in-use concentrations. Moreover, the times for bacterial complete killing were remarkably prolonged in DRS isolates even after a 10 min of exposure to 1000 mg/L of ADH, a half of its in-use concentration. The MICs of CHX for DRS isolates rose to 640 mg/L after repetitive passages in subinhibitory concentrations of CHX. CONCLUSIONS: Given that the bactericidal effects of the four major disinfectants were considerably reduced in the presence of organic material (BSA) and DRS isolates tended to adapt to CHX, continuous surveys of the profiles of susceptibility to disinfectants among clinically isolated Acinetobacter species are very necessary from the standpoint of nosocomial infection control.

Molecular characterization and expression of angiopoietin-like protein 3 in the chicken, Gallus gallus.

Angiopoietin-like protein 3 (ANGPTL3) is a 70 kDa secretory glycoprotein, which has been identified as a plasma triacylglycerol (TG)-increasing factor in KK/San mice. In this study, we have characterized the expression profile and activity of ANGPTL3 in the chicken, Gallus gallus. This is the first report of this protein in a pre-mammalian species and reveals novel details about the molecular structure of, and specific features of the expression of, important molecules in lipid metabolism. Recombinant chicken ANGPTL3 inhibited chicken lipoprotein lipase activity in vitro and increased plasma TG concentrations in vivo. Chicken ANGPTL3 mRNA expression was detected only in the liver. ANGPTL3 mRNA expression was significantly higher in mature (i.e. egg producing) chickens than in immature chickens. However, this increase was not associated with estradiol, although estradiol does enhance triacylglycerol and very low density lipoprotein secretion by the chicken liver. Changes in chicken ANGPTL3 mRNA expression with nutritional state were examined. ANGPLT3 expression was affected by certain nutritional treatments, i.e. fasting and re-feeding, and ANGPTL3 gene expression in the livers of chickens fed a cholesterol-supplemented diet was lower than that in the livers of chickens fed a control diet. The present study is the first to molecularly characterize and analyze the gene expression of ANGPTL3, and suggests that in avian species ANGPTL3 plays a key role in lipid delivery to the yolk.

Tamoxifen-induced Lung Injury.

Cases of drug-induced lung injury caused by tamoxifen are rare. A 74-year-old man underwent surgery for the treatment of right breast cancer; tamoxifen was administered as an adjuvant therapy after surgery. The patient developed cough and dyspnea and chest computed tomography showed ground glass opacification in the lower lobe of the right lung. He was diagnosed with tamoxifen-induced lung injury. The diagnosis was made based on the exclusion of other causes and recurrence with the re-administration of tamoxifen. Physicians should therefore be aware of the potential for the development of tamoxifen-induced lung injury.

Clinical significance of atypical squamous cells of undetermined significance after treatment for cervical intraepithelial grade 3 neoplasia: A retrospective single-center cohort study.

The aim of the present study was to evaluate the clinical significance of atypical squamous cells of undetermined significance (ASC-US) following cervical conization for cervical intraepithelial neoplasia (CIN) grade 3. This study was a retrospective cohort analysis. The medical records of women treated with conization for CIN 2-3 were reviewed and 142 patients with CIN 3 who had been diagnosed using the conization specimens were selected. The mean follow-up period after conization was 41.8 months. Cytological abnormalities after conization were observed in 19.0% of the patients and consisted of ASC-US (13.4%) and worse than low-grade squamous intraepithelial lesion (LSIL; 5.6%). Recurrence was defined as a diagnosis worse than CIN 2, and the recurrence rate was 29.6% among patients with abnormal cytology. The recurrence rate was 15.7% in the ASC-US group and 71.4% in the worse than LSIL group. There was no significant difference in the time of initial identification of abnormal cytology after treatment between the worse than LSIL and the ASC-US groups (P=0.054). However, the ASC-US group had a significantly better cumulative recurrence-free rate compared with the worse than LSIL group (P<0.05). Women with ASC-US following treatment for CIN appear to be at a relatively high risk. Regarding the risk stratification of women following treatment for CIN, if surveillance cytology shows ASC-US, immediate colposcopy is recommended, along with long-term follow-up.

Supramolecular complexes of La@C82 with unsaturated thiacrown ethers.

The paramagnetic La@C82-A(C2v) with unsaturated thiacrown ethers forms 1 : 1 host-guest complexes of [La@C82-A(C2v)]-[D]+ in solution as a result of electron transfer.

In vitro susceptibility and genetic variations for chloroquine and mefloquine in Plasmodium falciparum isolates from Thai-Myanmar border.

In vitro drug susceptibility to chloroquine (CQ) and mefloquine (MF) were assessed in 39 P. falciparum isolates from the Thai-Myanmar border area. To further characterize CQ- and MF-resistance profiles in this area, we also analyzed pfcrt K76T mutation that is critical for CQ resistance, and pfmdr1 polymorphism that has an association with MF resistance. Eighteen isolates were successfully examined by in vitro tests for CQ, and 17 of them had resistance to the drug. Geometric mean concentration of CQ that inhibited the growth of parasites at 50% (IC50) was 371 +/- 227 nM (105-971 nM). Sixteen isolates were successfully examined by in vitro tests for MF, and 8 of them were resistant to the drug. Geometric mean of IC50 for MF was 41 +/- 31 nM (4-125 nM). Genotypes of drug resistance, such as pfcrt and pfmdr1 mutations, were also analyzed. All the 39 isolates had the same haplotype (CVIET) for PfCRT at its 72-76th amino acids. A pfmdr1 Y86 mutation was found in 95% of isolates. A pfmdr1 D1042 mutation was also present in 7 isolates, while no pfmdr1 Y1246 mutation was observed. These results indicated a correlation between CQ resistance and the pfcrt T76 and pfmdr1 Y86 mutations.