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.

Anti-trypanosomal Lignans Isolated from Salvadoran Peperomia pseudopereskiifolia.

A search for anti-trypanosomal natural compounds from plants collected in El Salvador, a country particularly endemic for Chagas disease, resulted in the isolation of five lignan-type compounds (1-5) from Peperomia pseudopereskiifolia. The lignan derivatives 1, 2, and 4 are new. Their absolute configuration was determined by chemical derivatization. Compounds 1, 5, 6, and 8 exhibited anti-trypanosomal activity against the amastigote form of T. cruzi comparable to that of the existing drug benznidazole.

Compound-compound interaction analysis of baicalin and berberine derivatives in aqueous solution.

Baicalin and berberine are biologically active constituents of the crude drugs Scutellaria root and Coptis rhizome/Phellodendron bark, respectively. Baicalin and berberine are reported to combine together as a 1:1 complex that forms yellow precipitates by electrostatic interaction in decoctions of Kampo formulae containing these crude drugs. However, the structural basis and mechanism for the precipitate formation of this compound-compound interaction in aqueous solution remains unclarified. Herein, we searched for berberine derivatives in the Coptis rhizome that interact with baicalin and identified the chemical structures involved in the precipitation formation. Precipitation assays showed that baicalin formed precipitates with berberine and coptisine but not with palmatine and epiberberine. Thus, the 2,3-methylenedioxy structure may be crucial to the formation of the precipitates, and electrostatic interaction is necessary but is not sufficient. In this multicomponent system experiment, palmatine formed a dissociable complex with baicalin and may competitively inhibit the formation of berberine and coptisine precipitation with baicalin. Therefore, the precipitation formed by berberine and baicalin was considered to be caused by the aggregation of the berberine-baicalin complex, and the 2,3-methylenedioxy structure is likely crucial to the aggregation of the complex.

Capability of a large bacterial artificial chromosome clone harboring multiple biosynthetic gene clusters for the production of diverse compounds.

The biosynthetic gene clusters (BGCs) for the macrocyclic lactone-based polyketide compounds are extremely large-sized because the polyketide synthases that generate the polyketide chains of the basic backbone are of very high molecular weight. In developing a heterologous expression system for the large BGCs amenable to the production of such natural products, we selected concanamycin as an appropriate target. We obtained a bacterial artificial chromosome (BAC) clone with a 211-kb insert harboring the entire BGC responsible for the biosynthesis of concanamycin. Heterologous expression of this clone in a host strain, Streptomyces avermitilis SUKA32, permitted the production of concanamycin, as well as that of two additional aromatic polyketides. Structural elucidation identified these additional products as ent-gephyromycin and a novel compound that was designated JBIR-157. We describe herein sequencing and expression studies performed on these BGCs, demonstrating the utility of large BAC clones for the heterologous expression of cryptic or near-silent loci.

Physical Rehabilitation and Post-Stroke Pneumonia: A Retrospective Observational Study Using the Japanese Diagnosis Procedure Combination Database.

In this study, the relationship between the duration of physical rehabilitation and occurrence of pneumonia after ischemic stroke was examined. We included 426,508 patients aged ≥75 years with acute ischemic stroke. A multilevel logistic regression analysis nested at the hospital level was conducted to examine the association between the duration of physical rehabilitation and occurrence of pneumonia. The duration of physical rehabilitation refers to the hours of physical rehabilitation performed daily until the 7th day of hospitalization. In the multivariable analysis, the intensity of rehabilitation for durations of 20-39 min/day (adjusted odds ratio [aOR]: 0.78, 95% Confidence Interval [CI]: 0.75-0.81, p < 0.001), 40-59 min/day (aOR: 0.68, 95% CI: 0.66-0.71, p < 0.001), 60-79 min/day (aOR:0.56, 95% CI: 0.53-0.58, p < 0.001), and ≥80 min/day (aOR: 0.46, 95% CI: 0.44-0.48, p < 0.001) were significantly associated with a reduced incidence of pneumonia. In addition, the trend identified for duration of rehabilitation was significant (p < 0.001). The results of this study suggest the usefulness of high-duration physical rehabilitation for preventing pneumonia in older patients with ischemic stroke.

Atrial fibrillation and mortality after ischemic stroke: An observational study using an insurance claim database.

OBJECTIVES: In this study, we aimed to examine the association between atrial fibrillation and mortality after ischemic stroke and evaluate the use of anticoagulation therapy for atrial fibrillation before stroke onset in patients who experienced stroke. METHODS: In this retrospective observational study, we used a combined database of medical and long-term care insurance claims data from one prefecture in Japan. The data of 25,352 patients aged ≥ 65 years who were hospitalized in acute care hospitals with a diagnosis of ischemic stroke between April 2012 and March 2015 were extracted. Cox proportional hazard modeling, with adjustment for age, sex, comorbidities, and long-term care dependency level (based on the activities of daily living), was performed to evaluate the relationship between mortality and atrial fibrillation. RESULTS: The prevalence of atrial fibrillation was 21.8% in the study population. A significant association was noted between mortality and atrial fibrillation (adjusted hazard ratio: 1.28, 95% confidence interval: 1.16-1.41, p < 0.001). Anticoagulant drugs were used in 32.2% of the patients with atrial fibrillation. CONCLUSIONS: These results indicate that atrial fibrillation is associated with mortality after stroke; however, the use of anticoagulation therapy for atrial fibrillation is unsatisfactory. Efforts to improve the use of atrial fibrillation therapy are required in Japan.

Association between early diagnosis of and inpatient mortality from invasive pulmonary aspergillosis among patients without immunocompromised host factors: a nationwide observational study.

OBJECTIVES: The incidence of invasive pulmonary aspergillosis (IPA) among patients without immunocompromised host factors (ICHF) has been described extensively. However, its diagnosis remains challenging. To date, no study has statistically confirmed the efficacy of early IPA diagnosis in patients without ICHF. METHODS: We conducted a cross-sectional study on mortality from IPA among patients without ICHF, using the Japanese Diagnosis Procedure Combination National Inpatient Database (April 2014-March 2018). The early diagnosis group was defined according to antifungal therapy initiation within 7 days of hospital admission. The delayed diagnosis group was defined according to antifungal therapy initiation between 8 and 28 days of the hospitalization. Associations were estimated using multivariate logistic regression. RESULTS: A total of 423 patients were registered (early diagnosis group, n = 262, 62%). The early diagnosis group had a lower mortality rate (30%) than the delayed diagnosis group (42%). The early diagnosis group that was treated with voriconazole was associated with lower odds of mortality (odds ratio 0.55, 95% confidence interval 0.31-0.99, P = 0.047). An age of ≥65 years and mechanical ventilation were associated with a higher mortality rate. CONCLUSION: Early diagnosis along with optimal antifungal treatment are crucial for achieving favorable outcomes among patients with IPA without ICHF.

Hemiacetal-less rapamycin derivatives designed and produced by genetic engineering of a type I polyketide synthase.

Engineering polyketide synthases is one of the most promising ways of producing a variety of polyketide derivatives. Exploring the undiscovered chemical space of this medicinally important class of middle molecular weight natural products will aid in the development of improved drugs in the future. In previous work, we established methodology designated 'module editing' to precisely manipulate polyketide synthase genes cloned in a bacterial artificial chromosome. Here, in the course of investigating the engineering capacity of the rapamycin PKS, novel rapamycin derivatives 1-4, which lack the hemiacetal moiety, were produced through the heterologous expression of engineered variants of the rapamycin PKS. Three kinds of module deletions in the polyketide synthase RapC were designed, and the genetically engineered vectors were prepared by the in vitro module editing technique. Streptomyces avermitilis SUKA34 transformed with these edited PKSs produced new rapamycin derivatives. The planar structures of 1-4 established based on 1D and 2D NMR, ESI-TOF-MS and UV spectra revealed that 2 and 3 had skeletons well-matched to the designs, but 1 and 4 did not. The observations provide important insights into the mechanisms of the later steps of rapamycin skeletal formation as well as the ketone-forming oxygenase RapJ.

JBIR-155, a Specific Class D ß-Lactamase Inhibitor of Microbial Origin.

We discovered JBIR-155 as a novel specific class D ß-lactamase inhibitor from Streptomyces polymachus SoB100815Hv02. JBIR-155 consists of a 6-oxabicyclo[3.2.0]heptan-7-one skeleton and a long unsaturated alkyl chain moiety of which absolute configuration was determined by spectroscopic data, modified Mosher's method, and analyses of the relative configuration of chemically modified derivative. JBIR-155 specifically exhibited inhibitory activity against the class D ß-lactamase, with an IC50 value of 0.36 µM.

A novel oxazole-containing tetraene compound, JBIR-159, produced by heterologous expression of the cryptic trans-AT type polyketide synthase biosynthetic gene cluster.

Using genome mining approach, we identified a novel biosynthetic gene cluster containing trans-AT type PKS genes from Streptomyces versipellis 4083-SVS6. A bacterial artificial chromosome (BAC) clone, pKU503JL68_PN1_P10-C12, accommodating the entire biosynthetic gene cluster was obtained from a BAC library. Heterologous expression of the biosynthetic gene cluster in Streptomyces lividans TK23 led to the production of a novel polyene compound, JBIR-159. We report herein the biosynthetic gene cluster for JBIR-159, and the heterologous expression, isolation, structure determination and a brief biological activity.

In vitro Cas9-assisted editing of modular polyketide synthase genes to produce desired natural product derivatives.

One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.

Tibial shape and size predicts the risk of tibial plateau fracture after cementless unicompartmental knee arthroplasty in Japanese patients.

AIMS: Cementless unicompartmental knee arthroplasty (UKA) has advantages over cemented UKA, including improved fixation, but has a higher risk of tibial plateau fracture, particularly in Japanese patients. The aim of this multicentre study was to determine when cementless tibial components could safely be used in Japanese patients based on the size and shape of the tibia. METHODS: The study involved 212 cementless Oxford UKAs which were undertaken in 174 patients in six hospitals. The medial eminence line (MEL), which is a line parallel to the tibial axis passing through the tip of medial intercondylar eminence, was drawn on preoperative radiographs. Knees were classified as having a very overhanging medial tibial condyle if this line passed medial to the medial tibial cortex. They were also classified as very small if a size A/AA tibial component was used. RESULTS: The overall rate of fracture was 8% (17 out of 212 knees). The rate was higher in knees with very overhanging condyles (Odds ratio (OR) 13; p < 0.001) and with very small components (OR 7; p < 0.001). The OR was 21 (p < 0.001) in those with both very overhanging condyles and very small components. In all, 69% of knees (147) had neither very overhanging nor very small components, and the fracture rate in these patients was 1.4% (2 out of 147 knees). Males had a significantly reduced risk of fracture (OR 0.13; p = 0.002), probably because no males required very small components and females were more likely to have very overhanging condyles (OR 3; p = 0.013). 31% of knees (66) were in males and in these the rate of fracture was 1.5% (1 out of 66 knees). CONCLUSION: The rate of tibial plateau fracture in Japanese patients undergoing cementless UKA is high. We recommend that cemented tibial fixation should be used in Japanese patients who require very small components or have very overhanging condyles, as identified from preoperative radiographs. In the remaining 69% of knees cementless fixation can be used. This approach should result in a low rate of fracture. Cite this article: Bone Joint J 2020;102-B(7):861-867.

Removal of the E-Olefin Barrier of Humulene Leading to Unnatural Terpenoid-like Skeletons.

An unnatural terpenoid scaffold containing a bicyclo[5.4.0]undecane moiety, as well as a salvialane skeleton based on an intramolecular C-C bond formation strategy were synthesized. Such a strategy was made possible by the removal of strained E-olefin conformations of the humulene skeleton. Some compounds were identified to show PPARα antagonist activity.

Preparation of 1L-myo-Inositol 1-Phosphate as a Substrate of Phosphatidylinositol Phosphate Synthase.

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.

Development of a Terpenoid Alkaloid-like Compound Library Based on the Humulene Skeleton.

Many natural terpenoid alkaloid conjugates show biological activity because their structures contain both sp3 -rich terpenoid scaffolds and nitrogen-containing alkaloid scaffolds. However, their biosynthesis utilizes a limited set of compounds as sources of the terpenoid moiety. The production of terpenoid alkaloids containing various types of terpenoid moiety may provide useful, chemically diverse compound libraries for drug discovery. Herein, we report the construction of a library of terpenoid alkaloid-like compounds based on Lewis-acid-catalyzed transannulation of humulene diepoxide and subsequent sequential olefin metathesis. Cheminformatic analysis quantitatively showed that the synthesized terpenoid alkaloid-like compound library has a high level of three-dimensional-shape diversity. Extensive pharmacological screening of the library has led to the identification of promising compounds for the development of antihypolipidemic drugs. Therefore, the synthesis of terpenoid alkaloid-like compound libraries based on humulene is well suited to drug discovery. Synthesis of terpenoid alkaloid-like compounds based on several natural terpenoids is an effective strategy for producing chemically diverse libraries.

Quasi-stadium laser diodes with an unstable resonator condition.

We have observed lasing in a complicated eigenmode of a quasi-stadium laser diode with an unstable resonator consisting of two curved end mirrors obeying an unstable resonator condition and two straight sidewall mirrors. The laser was fabricated by application of a reactive-ion-etching technique to a molecular beam epitaxy-grown graded-index separate-confinement heterostructure single-quantum-well GaAs/AlGaAs structure. The far-field pattern shows that the lasing mode corresponds to the complicated lowest-loss mode obtained numerically by an extended Fox-Li method.

Lasing on scar modes in fully chaotic microcavities.

Scar wave functions in a fully chaotic cavity are obtained numerically by an extended Fox-Li method. Lasing on the scar modes are observed in a semiconductor microcavity with a selective excitation of different scars controlled by corresponding shape of electrodes for current injection.

Ring and axis mode lasing in quasi-stadium laser diodes with concentric end mirrors.

We fabricated quasi-stadium laser diodes whose resonators consist of two concentric curved end mirrors and two straight sidewall mirrors. We observed two lasing modes that correspond to different beam propagations along the cavity axis and along a ring trajectory, and different far-field patterns with wide angular separation. The modes can be selected by control of an electrode pattern. We also show that the far-field patterns numerically obtained by the extended Fox-Li mode calculation method are in good agreement with the experimental results.