Actinoplanes kirromycinicus sp. nov., isolated from soil.

A novel actinomycete, designated as TPMA0078T, was isolated from a soil sample collected in Shinjuku, Tokyo, Japan. 16S rRNA gene sequence analysis indicated that strain TPMA0078T belongs to the genus Actinoplanes and is closely related to Actinoplanes regularis IFO 12514T (99.86% 16S rRNA gene sequence similarity). The spores of strain TPMA0078T were motile, and the sporangia were cylindrical. The diamino acids in the cell wall peptidoglycan of strain TPMA0078T were meso-diaminopimelic acid and 3OH-meso-diaminopimelic acid. Whole-cell sugars were glucose and mannose, with galactose as a minor component. The major cellular fatty acids identified were iso-C15:0, iso-C16:0, and anteiso-C17:0. The predominant menaquinone was MK-9(H4), and the principal polar lipid was phosphatidylethanolamine. These chemotaxonomic properties of strain TPMA0078T were consistent with those of Actinoplanes. Meanwhile, digital DNA-DNA hybridization and average nucleotide identity values showed low relatedness between strain TPMA0078T and A. regularis NBRC 12514T. Furthermore, several phenotypic properties of strain TPMA0078T distinguished it from those of closely related species. Based on its genotypic and phenotypic characteristics, strain TPMA0078T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes kirromycinicus sp. nov. is proposed. The type strain is TPMA0078T (=NBRC 116422T = TBRC 18262T).

Isolation of Hydrazide-alkenes with Different Amino Acid Origins from an Azoxy-alkene-Producing Mutant of Streptomyces rochei 7434AN4.

A triple mutant (strain KA57) of Streptomyces rochei 7434AN4 produces an azoxy-alkene compound, KA57A, which was not detected in a parent strain or other single and double mutants. This strain accumulated several additional minor components, whose structures were elucidated. HPLC analysis of strain KA57 indicated the presence of two UV active components (KA57D1 and KA57D2) as minor components. They exhibited a maximum UV absorbance at 218 nm, whereas a UV absorbance of azoxy-alkene KA57A was detected at 236 nm, suggesting that both KA57D1 and KA57D2 contain a different chromophore from KA57A. KA57D1 has a molecular formula of C12H22N2O2, and NMR analysis revealed KA57D1 is a novel hydrazide-alkene compound, (Z)-N-acetyl-N'-(hex-1-en-1-yl)isobutylhydrazide. Labeling studies indicated that nitrogen Nß of KA57D1 is derived from l-glutamic acid, and the isobutylamide unit (C-1 to C-3, 2-Me, and Nα) originates from valine. KA57D2 has a molecular formula of C13H24N2O2, and its structure was determined to be (Z)-N-acetyl-N'-(hex-1-en-1-yl)-2-methylbutanehydrazide, in which a 2-methylbutanamide unit was shown to originate from isoleucine. Different biogenesis of the Nα atom (l-serine for KA57A, l-valine for KA57D1, and l-isoleucine for KA57D2) indicates the relaxed substrate recognition for nitrogen-nitrogen bond formation in the biosyntheses of KA57A, KA57D1, and KA57D2.

Isolation, Biosynthetic Investigation, and Biological Evaluation of Maniwamycin G, an Azoxyalkene Compound from Streptomyces sp. TOHO-M025.

A new maniwamycin analogue, maniwamycin G, was isolated from Streptomyces sp. TOHO-M025 as a major product. Maniwamycin G has a molecular formula of C12H22N2O4, and its extensive NMR analysis revealed that maniwamycin G contains a methoxycarbonyl group instead of an amide as found in maniwamycin F. Its C-2 and C-3 configurations were determined to be (2R, 3R) by circular dichroism spectrum and a modified Mosher method, respectively. The biosynthetic origin of maniwamycin G was investigated using isotope-labeled compounds. The carbon source of maniwamycin G is four acetate units (C-1', C-2'; C-3', C-4'; C-5', C-6'; and C-4, C-5) and l-serine (C-1 to C-3). The nitrogen atom attached at C-2 (Nα) originates from serine, whereas the nitrogen atom of a hexen-1-yl amine unit (Nß) is derived from glutamic acid. The quorum-sensing inhibitory activity of maniwamycin G was 2-fold lower than that of maniwamycin F.

Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450.

MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.

Actinocatenispora comari sp. nov., an endophytic actinomycete isolated from aerial parts of Comarum salesowianum.

A novel actinomycete, designated NUM-2625T, was isolated as an endophytic bacterium in aerial parts of Comarum salesowianum, an endemic species in the Altai, Himalaya mountain chain area, collected from Khasagt Khairkhan Mountain in Mongolia. The 16S rRNA gene sequence of strain NUM-2625T showed the highest similarity to Actinocatenispora thailandica TT2-10T (99.4 %), Actinocatenispora sera KV-744T (99.3 %), and Actinocatenispora rupis CS5-AC17T (97.7 %). Chemotaxonomic properties of strain NUM-2625T were essentially consistent with those of the genus Actinocatenispora, such as the presence of meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, MK-9(H4) and MK-9(H6) as the major menaquinones, and iso-C16 : 0, iso-C15 : 0, iso-C14 : 0 3-OH, and anteiso-C17 : 0 as the major fatty acids. Meanwhile, digital DNA-DNA hybridization and average nucleotide identity values revealed a low relatedness between strain NUM-2625T and the other type strains of the genus Actinocatenispora. In addition, strain NUM-2625T exhibited several phenotypic properties that could be used to distinguish it from its closest relatives. Based on the results of polyphasic analyses, strain NUM-2625T represents a novel species in the genus Actinocatenispora, for which the name Actinocatenispora comari sp. nov. is proposed. The type strain is NUM-2625T (=NBRC 114660T=TBRC 13496T).

Artificial control of the multistep oxidation reactions catalyzed by the cytochrome P450 enzyme RosC.

The cytochrome P450 monooxygenase RosC catalyzes the three-step oxidation reactions, which leads to the formation of a hydroxy, formyl, and carboxy group at C-20 during rosamicin biosynthesis in Micromonospora rosaria IFO13697. To determine if amino acid substitutions in RosC could allow for the control of the multistep oxidation reactions, we screened RosC random mutants. The RosC mutant RM30, with five amino acid substitutions (P107S, L176Q, S254N, V277A, and I319N), catalyzed only the first step of the oxidation reaction. Whole-cell assays using Escherichia coli cells expressing RosC mutants with single and double amino acid substitutions derived from RM30 indicated that P107S/L176Q, P107S/V277A, P107S/I319N, L176Q/V277A, L176Q/I319N, and S254N/V277A significantly reduced the catalytic activity of the second reaction, which is alcohol oxidation. Of the previously mentioned mutants, double mutants containing L176Q, which was presumed to occur in the FG loop region, lost the total catalytic activity of the third reaction (aldehyde oxidation). Additionally, an engineered M. rosaria strain with rosC disruption, which introduced the gene encoding the RosC mutants P107S/L176Q and P107S/V277A preferentially produced 20-dihydrorosamicin, which is formed after the first oxidation reaction of RosC.

Quorum Sensing Inhibitors against Chromobacterium violaceum CV026 Derived from an Actinomycete Metabolite Library.

Quorum sensing (QS) is a microbial signaling system that regulates the expression of many virulence genes. Herein, we studied five compounds-No. 1: (E)-2-methyl-3- (4-nitro-phenyl)-acrylaldehyde; No. 29-2: pimprinine [5-(1H-indol-3-yl)-2-methyloxazole]; No. 48: (2E,4E)-2-methyl-5-phenyl-2,4-pentadienoic acid; No. 74: (3E,5E)-5-methyl-6-(4-nitrophenyl)-hexa-3,5-dien-2-ol; and No. 130: methyphenazine-1-carboxylate-derived from an actinomycete metabolite library. These compounds were confirmed to be QS inhibitors that reduced violacein production in Chromobacterium violaceum CV026. Additionally, compounds No. 1, No. 74, and No. 130 significantly reduced fluorescent pigment production in Pseudomonas aeruginosa ATCC 27853.

Combined Approach Versus 2 Conventional Approaches in Ultrasound-Guided Central Venous Catheterization: A Randomized Controlled Trial.

OBJECTIVE: The authors compared the occurrence of posterior wall puncture using the short-axis out-of-plane and long-axis in-plane approaches with that using the combined short-axis-and-long-axis approach that the authors previously showed to be effective in observational and manikin studies. DESIGN: Randomized controlled study. SETTING: Single tertiary institution. PARTICIPANTS: One hundred twenty patients who underwent cardiac or vascular surgery under general anesthesia. INTERVENTIONS: The patients were divided randomly into combined short-axis-and-long-axis (n = 40), short-axis out-of-plane (SA-OOP) (n = 40), and long-axis in-plane (LA-IP) (n = 40) groups and received ultrasound-guided central venous catheterization at the right internal jugular vein. MEASUREMENTS AND MAIN RESULTS: Successful guidewire insertion without posterior wall puncture was performed in 40 patients (100%) in the combined short-axis-and-long-axis approach group, 28 (70%) in the short-axis out-of-plane approach group, and 38 (95%) in the LA-IP approach group (combined short-axis-and-long-axis v SA-OOP, p = 0.0002 [relative risk = 1.43; 95% CI: 1.17-1.75]; combined short-axis-and-long-axis v LA-IP, p = 0.49 [relative risk = 1.05; 95% CI: 0.98-1.13]). Procedure durations were 28.5 (24.1-36.4) seconds in the combined short-axis-and-long-axis group, 31.7 (24.4-40.6) seconds in the SA-OOP group, and 24.3 (20.8-32.1) seconds in the long-axis in-plane group (combined short-axis-and-long-axis v SA-OOP, p = 0.53; combined short-axis-and-long-axis v LA-IP, p = 0.044). CONCLUSION: The combined short-axis-and-long-axis approach for ultrasound-guided central venous catheterization had a lower posterior wall puncture rate than the SA-OOP approach, but there was no significant difference with the long-axis in-plane approach.

Impact of concomitant tricuspid regurgitation on long-term outcomes in severe aortic stenosis.

AIMS: Tricuspid regurgitation (TR) has been reported to be associated with worse survival in various heart diseases, but there are few data in aortic stenosis (AS). METHODS AND RESULTS: In the Contemporary Outcomes after Surgery and Medical Treatment in Patients with Severe Aortic Stenosis (CURRENT AS) Registry enrolling 3815 consecutive patients with severe AS, there were 628 patients with moderate or severe TR (TR group) and 3187 patients with no or mild TR (no TR group). The study patients were subdivided into the initial aortic valve replacement (AVR) stratum (n = 1197) and the conservative stratum (n = 2618) according to treatment strategy. The primary outcome measure was a composite of aortic valve-related death or hospitalization due to heart failure. The 5-year freedom rate from the primary outcome measure was significantly lower in the TR group than in the no TR group (49.1% vs. 67.3%, P < 0.001). Even after adjusting for confounders, the excess risk of TR relative to no TR for the primary outcome measure remained significant [hazard ratio (HR): 1.25, 95% confidence interval (CI): 1.06-1.48; P = 0.008]. The trend for the excess adjusted risk in the TR group was consistent in the initial AVR and the conservative strata (HR 1.55, 95% CI: 0.97-2.48; P = 0.07; HR 1.22, 95% CI: 1.02-1.46; P = 0.03, respectively). In the initial AVR stratum, the 5-year freedom rate from the primary outcome measure was not different between the two groups with (n = 56) or without (n = 91) concomitant tricuspid annuloplasty (61.5% vs. 72.1%, P = 0.48). CONCLUSION: The presence of clinically significant TR concomitant with severe AS is associated with a poor long-term outcome, regardless of the initial treatment strategy.

Comparing Combined Short-Axis and Long-Axis Ultrasound-Guided Central Venous Catheterization With Conventional Short-Axis Out-of-Plane Approaches.

OBJECTIVE: Visualizing the needle tip using the short-axis out-of-plane (SA-OOP) ultrasound-guided central venous catheterization approach is difficult and results in posterior wall puncture (PWP). To improve needle tip visualization in the long-axis view, combining the SA-OOP and the long-axis in-plane approaches has been suggested. The authors, who previously reported on the utility of this technique using a manikin model, examined the feasibility of this novel method (referred to as the combined short-axis and long-axis [CSLA] approach) and compared the CSLA approach with the SA-OOP approach in humans for the present study. DESIGN: Prospective observational study. SETTING: Single institution, Rakuwakai Otowa Hospital. PARTICIPANTS: Patients undergoing cardiac or vascular surgeries. INTERVENTIONS: The CSLA and SA-OOP approaches were used for ultrasound-guided right jugular venous puncture. The puncturing procedures were determined arbitrarily preoperatively without consideration of the patient's neck anatomy and were based on the operator's preference without randomization. MEASUREMENTS AND MAIN RESULTS: The study comprised 100 patients. Successful guidewire insertion without PWP was performed in 48 patients (96%) in the CSLA approach group and 33 (66%) in the SA-OOP approach group; the rate was significantly higher in the CSLA approach group (p = 0.0001). The procedural durations were 27.5 (range 17.0-122.0) seconds in the CSLA approach group and 25.0 (range 15.0-158.0) seconds in the SA-OOP approach group (p = 0.19). CONCLUSIONS: This study showed that the CSLA approach to ultrasound-guided central venous catheterization might help prevent PWP.

Analysis of crosstalk-free conditions for a cross-shift multiplexing method in holographic data recording.

There are various multiplexing methods in holographic data recording. A larger storage capacity is anticipated when combining two different multiplexing methods and then ensuring crosstalk-free recording. We have proposed a cross-shift multiplexing method that combines shift multiplexing and peristrophic multiplexing. Crosstalk-free conditions for shift multiplexing and peristrophic multiplexing were separately clarified. However, these conditions cannot be individually applied to cross-shift multiplexing. Then, we deduced the analytic formula of crosstalk for cross-shift multiplexing and verified its validity by comparing the theoretical and experimental results. Using this analytic formula, the shift pitch and rotation angle that attain crosstalk-free multiplexing can be determined.

Early Surgery vs. Surgery After Watchful Waiting for Asymptomatic Severe Aortic Stenosis.

BACKGROUND: There is no large-scale study comparing postoperative mortality after aortic valve replacement (AVR) for asymptomatic severe aortic stenosis (AS) between initial treatment with AVR vs. eventual AVR after conservative management. Methods and Results: We analyzed data from a multicenter registry enrolling 3,815 consecutive patients with severe AS. Of 1,808 asymptomatic patients, 286 patients initially underwent AVR (initial AVR group), and 377 patients were initially managed conservatively and eventually underwent AVR (AVR after watchful waiting group). Mortality after AVR was compared between the 2 groups. Subgroup analysis according to peak aortic jet velocity (Vmax) at diagnosis was also conducted. There was no significant difference between the 2 groups in 5-year overall survival (OS; 86.0% vs. 84.1%, P=0.34) or cardiovascular death-free survival (DFS; 91.3% vs. 91.1%, P=0.61), but on subgroup analysis of patients with Vmax ≥4.5 m/s at diagnosis, the initial AVR group was superior to the AVR after watchful waiting group in both 5-year OS (88.4% vs. 70.6%, P=0.003) and cardiovascular DFS (91.9% vs. 81.7%, P=0.023). CONCLUSIONS: Asymptomatic severe AS patients who underwent AVR after watchful waiting had a postoperative survival rate similar to those who initially underwent AVR. In a subgroup of patients with Vmax ≥4.5 m/s at diagnosis, however, the AVR after watchful waiting group had worse postoperative survival rate than the initial AVR group.

Tracheal injury detected immediately after median sternotomy by inexperienced surgeons: two case reports.

BACKGROUND: Although median sternotomy is standard during cardiac surgery, the procedure is associated with a risk of injury to mediastinal organs. Here, we discuss two cases of tracheal injury following median sternotomy during cardiac surgery. CASE PRESENTATION: Ventilation failure occurred in a 78-year-old Japanese man and a 71-year-old Japanese man after median sternotomy, and tracheal injury was identified. The sites of injury were directly repaired and covered with mediastinal fat tissue, following which ventilation was successful. The burn-like deposits observed at the site of tracheal injury and on the removed endotracheal tube support the notion that the injuries in our patients were caused by electrocautery prior to median sternotomy. In one case, short sternotracheal distance may have contributed to tracheal injury during post-sternal manipulation. In both cases, the relative inexperience of both surgeons also supports the suspected cause of injury. CONCLUSIONS: Tracheal injury represents a potential complication following median sternotomy, especially when performed by inexperienced surgeons or in cases of short sternotracheal distance. Anesthesiologists should consider this rare yet potentially lethal complication.

Cytochrome P450 enzyme RosC catalyzes a multistep oxidation reaction to form the non-active compound 20-carboxyrosamicin.

The cytochrome P450 enzyme RosC catalyzes a two-step, hydroxylation and alcohol oxidation, oxidation reaction to form the C-20 formyl group in the biosynthesis of a 16-membered macrolide antibiotic rosamicin produced by Micromonospora rosaria IFO13697. RosC is presumed to be involved in the formation of 20-carboxyrosamicin because it has been isolated from the culture broth of M. rosaria. Here, we confirmed that RosC has catalytic activity, with E. coli expressing RosC converting rosamicin into 20-carboxyrosamicin. Therefore, it was revealed that RosC is a multifunctional P450 that catalyzes a three-step oxidation reaction that leads to the formation of the hydroxyl group, formyl group and carboxyl group at C-20 on the macrolactone ring in the rosamicin biosynthetic pathway. Moreover, the cytochrome P450 enzyme TylI, which is involved in formation of the formyl group of a 16-membered macrolide antibiotic tylosin produced by Streptomyces fradiae ATCC 19609, also converted rosamicin into 20-carboxyrosamicin.

Naphthacemycins, novel circumventors of ß-lactam resistance in MRSA, produced by Streptomyces sp. KB-3346-5. II. Structure elucidation.

Seventeen new compounds, naphthacemycins A1-A11, B1-B4 and C1-C2, were isolated from a cultured broth of Streptomyces sp. KB-3346-5 during screening for circumventors of ß-lactam resistance in methicillin-resistant Staphylococcus aureus. Their structures were elucidated by spectroscopic studies, including NMR and X-ray crystallographic analysis. The naphthacemycin A series has a new skeleton displaying a 7-phenylnaphthacene-5,6,11(12H)-trione. In contrast, the quinone moiety of the A series is changed to dehydroxyquinol in the B series and to a semiquinone-like structure in the C series.

Naphthacemycins, novel circumventors of ß-lactam resistance in MRSA, produced by Streptomyces sp. KB-3346-5. I. The taxonomy of the producing strain, and the fermentation, isolation and antibacterial activities.

Screening for circumventors of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) led us to find 17 novel antibiotics, naphthacemycins A1-A11, B1-B4 and C1-C2. The naphthacemycins were isolated from a cultured broth of Streptomyces sp. KB-3346-5 by repeated silica gel column chromatography and HPLC. Naphthacemycins enhanced imipenem activity 100-500 times against MRSA at 0.5 µg ml-1, and naphthacemycins A4-A11 themselves showed MIC50 values of 1-4 µg ml-1 against 22 MRSA strains.

Novel Technique Using Polyester Fabric and Fibrin Sealant Patch for Acute Aortic Dissection.

We describe a simple and effective technique for acute aortic dissection using a combination of polyester fabric and a fibrin sealant patch (FSP) to achieve effective reinforcement and haemostasis of the aortic stump. Firstly, the 0.61mm thick knitted polyester fabric sheet was cut to half of the size of the FSP. Next, fibrin glue was sprayed onto the collagen layer of the FSP. Subsequently, a fabric sheet was placed upon it, and the FSP was put together with the irrigated collagen layer, and then completely dried to bind the patch. As a result, the dry fibrinogen/thrombin layers, as an adhesive surface, faced outward. This patch was trimmed to a 10-15-mm-wide strip. The composite patch was inserted into the false lumen. The stump was gently pressed to fix the aortic intima and adventitia. There are several advantages: the combined patch can be prepared during systemic cooling, and therefore can minimise the circulatory arrest time; secondly, the false lumen is not directly exposed to fibrin glue and so the risk of embolism is extremely low; thirdly, the expected haemostatic effect is greater as FSP lines the exterior of the intima, achieving haemostasis for suture holes.

Maniwamycins: new quorum-sensing inhibitors against Chromobacterium violaceum CV026 were isolated from Streptomyces sp. TOHO-M025.

Quorum sensing is an important microbial signaling system that controls the expression of many virulence genes. Maniwamycins C-F, new compounds and quorum-sensing inhibitors, were isolated from the culture broth of Streptomyces sp. TOHO-M025 using a silica gel column and preparative HPLC. The structures of maniwamycins were elucidated by spectroscopic analyses, including NMR. The compounds each have an azoxy moiety. All maniwamycins inhibited violacein synthesis, which is controlled by quorum sensing, in Chromobacterium violaceum CV026.

Direct thiophenylation accompanying orthoester-cleavage of 1,2,4-O-orthoacetyl-3,6-O-(o-xylylene)glucopyranose.

The 3,6-O-(o-xylylene) bridge locks the conformation of glucopyranose to an axial-rich form. Although the conformational lock induces complete ß-selectivity in a glycosylation reaction, the leaving group of the glycosyl donor is limited to fluorine. On the other hand, the bridge confers the furanose-preferred property to glucose, which makes synthesis of corresponding pyranosyl derivatives that equip various leaving groups difficult. This problem was solved through direct phenylthio glucosidation of 3,6-O-(o-xylylene)-1,2,4-O-orthoacetylglucose accompanying cleavage of the orthoester moiety. This paper describes the process of establishing direct thiophenylation. This process reduced the synthetic steps for the known glucopyranosyl fluoride and will expand application of conformationally locked glycosyl donors.

A new mycinosyl rosamicin derivative produced by an engineered Micromonospora rosaria mutant with a cytochrome P450 gene disruption introducing the D-mycinose biosynthetic gene.

Genetic engineering of post-polyketide synthase-tailoring genes can be used to generate new macrolide analogs through manipulation of the genes involved in their biosynthesis. Rosamicin, a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697, contains a formyl group and an epoxide at C-20 and C-12/13 positions which are formed by the cytochrome P450 enzymes RosC and RosD, respectively. The D-mycinose biosynthesis genes in mycinamicin II biosynthesis gene cluster of Micomonospora guriseorubida A11725 were introduced into the rosC and rosD disruption mutants of M. rosaria IFO13697. The resulting engineered strains, M. rosaria TPMA0054 and TPMA0069, produced mycinosyl rosamicin derivatives, IZIV and IZV, respectively. IZIV was identified as a novel mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydrorosamicin.