A novel carotenoid biosynthetic route via oxidosqualene.

Over 800 known carotenoids are synthesized from phytoene or 4,4'-diapophytoene (dehydrosqualene) characterized by three conjugated double bonds. In this paper, we report that carotenoid desaturase CrtN from Staphylococcus aureus and Methylomonas can accept oxidosqualene, which is the precursor for plant- or animal-type triterpenoids, yielding the yellow carotenoid pigments with 8, 9, or 10 conjugated double bonds. The resulting pathway is the second nonnatural route for carotenoid pigments and the first pathway for carotenoid pigments not biosynthesized via (diapo)phytoene.

Severe epidemic myalgia with an elevated level of serum interleukin-6 caused by human parechovirus type 3: a case report and brief review of the literature.

BACKGROUND: Human parechovirus type 3 (HPeV-3) is known to cause cold-like symptoms, diarrhea, or severe infections such as sepsis in infants and children. In adults, HPeV-3 infection is rarely diagnosed because the symptoms are generally mild and self-limiting; however, this infection has been linked to epidemic myalgia, regardless of the presence of underlying diseases, immunosuppression, or sex. CASE PRESENTATION: We describe an adult case of severe systemic myalgia and orchiodynia after infection with HPeV-3, which was transmitted from the child of the patient. Interleukin-6 (IL-6) level was found to be elevated in the patient's serum. CONCLUSION: Severe myalgia associated with HPeV-3 infection is potentially caused by an elevated serum level of IL-6.

Tweezing the cofactor preference of gymnosperm pinene synthase.

The cellular activities of gymnosperms monoterpene synthases are largely compromised due to their requirement for manganese, which is deficient in microbial cells. Through site-saturation mutagenesis of the residue adjacent to metal-binding glutamate, we found that pinene synthase is highly mutable at this position yet drastically alter their metal binding preference, thereby quickly improving the cellular performance in heterologous hosts.

Preparation of Microvolume Anion-Exchange Cartridge for Inductively Coupled Plasma Mass Spectrometry-Based Determination of (237)Np Content in Spent Nuclear Fuel.

Microvolume anion-exchange porous polymer disk-packed cartridges were prepared for Am/Np separation, which is required prior to the measurement of Neptunium-237 ((237)Np) with inductively coupled plasma mass spectrometry (ICPMS). Disks with a volume of 0.08 cm(3) were cut out from porous sheets having anion-exchange-group-containing polymer chains densely attached on the pore surface. Four different amine-based groups, N,N-dimethylaminoethyl methacrylate, trimethylammonium, diethylamine, and triethylenediamine (TEDA), were selected as the anion-exchange groups to be introduced into the porous sheets. The separation performances of Am/Np were evaluated using a standard solution of (243)Am, which had the same activity as its daughter nuclide (239)Np in secular equilibrium. (239)Np recovery of close to 100% with practically no contamination of (243)Am was achieved using the TEDA-introduced disk-packed cartridge. The time to elute (239)Np from the cartridge was approximately 40 s. The TEDA-introduced disk-packed cartridge was applied to the separation of Np from a spent nuclear fuel sample to confirm its separation performance. A known amount of (243)Am ((239)Np) was added to the spent nuclear fuel sample solution to monitor the chemical yield of Np. The chemical yield of Np calculated from a measured concentration of (239)Np was 90.4%. Am leakage in the Np-eluted solution was less than 1 ppt, corresponding to 0.001% of the original Am concentration in the sample. This indicates that no additional (239)Np was produced by the decay of the (243)Am remaining in the Np-eluted solution, thus providing a reliable chemical yield. U, which can cause a serious spectral interference involving the peak tail from the mass spectrum of (238)U, was thoroughly removed with the TEDA cartridge, providing interference-free measurement of (237)Np. The concentration of (237)Np obtained by ICPMS was 718 ± 12 ng/mg-U, which agrees well with the theoretically calculated value. Compared with the conventional separation technique using commercially available anion-exchange resin columns, the time required to adsorb, wash, and elute Np using the TEDA- introduced disk-packed cartridge was reduced by 75%.

A Multicenter Study on the Utility of Selective Enrichment Broth for Detection of Group B Streptococcus in Pregnant Women in Japan.

Universal screening for Streptococcus agalactiae, Group B Streptococcus (GBS), in pregnant women is important for the prevention of severe infectious diseases in neonates. The subculture method using selective enrichment broth significantly improves GBS detection rates in the United States; however, this method is not widely utilized in Japan mainly because of the lack of large-scale validation. Therefore, we aimed to validate the utility of the subculture method in collaboration with multiple facilities. A total of 1957 vaginal-rectal swab specimens were obtained from pregnant women at 35-37 gestational weeks from March 1, 2020, to August 30, 2020, at Fukushima Medical University Hospital, Aiiku Hospital, Kitano Hospital, and the University of the Ryukyus Hospital. Conventional direct agar plating, subculture using selective enrichment broth, and direct latex agglutination (LA) testing with incubated broth were performed for GBS detection, and discrepant results were confirmed using real-time PCR. The GBS detection rates for direct agar plating, subculture, and direct LA testing were 18.2% (357/1957), 21.6% (423/1957), and 22.3% (437/1957), respectively. The use of selective enrichment broth showed promise for GBS detection with high sensitivity and is therefore recommended for GBS screening to prevent GBS-related infectious diseases in neonates in Japan.

In vitro activity of tedizolid against 43 species of Nocardia species.

The purpose of the present study was to evaluate the in vitro activity of tedizolid against several clinically significant species of Nocardia by comparing with that of linezolid. A total of 286 isolates of Nocardia species, including 236 clinical isolates recovered from patients in Japan and 50 strains (43 species) purchased from NITE Biological Resource Center, were studied. Antimicrobial susceptibility testing was performed using the broth microdilution method. For the 286 Nocardia isolates, the minimal inhibitory concentration (MIC)50 and MIC90 values of tedizolid were 0.25 and 0.5 µg/ml, and those of linezolid were 2 and 2 µg/ml, respectively. The distribution of the linezolid/tedizolid ratios (MICs of linezolid/MICs of tedizolid) showed that tedizolid had four- to eight-fold higher activity than linezolid in 96.1% (275/286) of Nocardia isolates. Both the tedizolid and linezolid MIC90 values for Nocardia brasiliensis were two-fold higher than those for the other Nocardia species. Both tedizolid and linezolid had low MIC values, 0.25-1 µg/ml and 0.5-4 µg/ml, respectively, even against nine isolates (five species) that were resistant to trimethoprim/sulfamethoxazole. One Nocardia sputorum isolate showed reduced susceptibility to tedizolid (4 µg/ml). Bioinformatics analysis suggests different resistance mechanisms than the oxazolidinone resistance seen in enterococci and staphylococci.

Immobilization of an esterase inhibitor on a porous hollow-fiber membrane by radiation-induced graft polymerization for developing a diagnostic tool for feline kidney diseases.

Removal of the major urinary protein, cauxin, a carboxylesterase, from cat urine is essential for distinguishing between physiological and abnormal proteinuria by a urine dipstick. We have previously developed a material for removing cauxin by using lens culinaris agglutinin (LCA) lectin which targets the N-linked oligosaccharides present in cauxin. To improve the affinity and specificity toward cauxin, we immobilized 1,1,1-trifluoro-3-(2-sulfanylethylsulfanyl) propane-2-one, an inhibitor of esterases, to a polymer chain grafted on to a porous hollow-fiber membrane by applying radiation-induced graft polymerization. Normal male urine was forced to permeate through the pores rimmed by the ligand-immobilized polymer chain. Cauxin could not be detected in the effluent from the membrane. The residence time of the urine across a membrane thickness of 1 mm was set at 7 s. The respective dynamic and equilibrium binding capacities of the membrane for cauxin were 2 and 3 mg/g. The developed cauxin-affinity membrane material was more effective for diagnosing cat kidney diseases than the LCA lectin tip.

A nucleoside kinase as a dual selector for genetic switches and circuits.

The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecedented enrichment efficacy (>30,000-fold) of functional switches from non-functional ones in a single selection cycle. In addition, negative (OFF) selection was exceptionally stringent, allowing the rapid and efficient selection of non-leaky from leaky circuits.

1H NMR assignment of oligomeric grafts of maleic anhydride-grafted polyolefin.

The (1)H NMR assignment of oligomeric grafts of maleic anhydride (MA)-grafted polyolefin (PO), MA-g-PO hereafter, was experimentally demonstrated for the first time using NMR spectroscopy. (13)C DEPT, (1)H-(1)H DQF-COSY, and (1)H T(2)-edited spectroscopy of MA-g-PO proved that peaks of the intermediate methine protons of succinic anhydride oligomeric grafts, which are nearly tetrameric, are observed at 2.5-3.5 ppm and show broadening.

Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry with Time-of-Flight Peak Analysis for Rapid and Accurate Detection of Group B Streptococcus in Pregnant Women.

Severe infections in neonates caused by Streptococcus agalactiae, Group B Streptococcus (GBS), are often associated with GBS transmission from their mothers during labor or birth. Hence, it is necessary to develop a universal method for screening vaginal-rectal GBS colonization in pregnant women worldwide. A subculture of vaginal-rectal swabs using a selective enrichment broth and an agar plate is conventionally recommended for GBS screening. However, infants born to mothers who are GBS negative on subculture sometimes contract GBS infections. Therefore, we developed another method with high sensitivity for GBS screening. A total of 178 vaginal-rectal swabs from pregnant women were inoculated into the enrichment broth, of which 126 were suspected of containing GBS due to the change in the color of the broth. The subculture results were positive for GBS in 34 (27.0%) swabs. Each broth was then analyzed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Analysis of the TOF peaks specific to GBS revealed 45 (35.7%) swabs as GBS positive. Of the 11 GBS positive samples on TOF peak analysis but negative on subculture, S. agalactiae gene targets were detected through PCR in 4 samples. MALDI detection with analysis of peaks of TOF (MDAPT) can detect GBS directly from cultured broth with high sensitivity. MDAPT can be an alternative method for GBS screening in pregnant women and contribute to the prevention of severe GBS infectious diseases in neonates. IMPORTANCE As previously reported, 10%-30% of pregnant women carry Streptococcus agalactiae, Group B Streptococcus (GBS), in their vagina or rectum, and approximately 50% of them vertically transmit GBS to their neonates during labor or birth. Moreover, 1%-2% of the GBS-transmitted neonates develop severe GBS infectious diseases, which have a mortality rate of 19.2% in a preterm infant and 2.1% in a full-term infant. Hence, universal screening for GBS colonization in pregnant women is conducted worldwide using the subculture procedure; however, infants born to GBS negative mothers sometimes contract GBS infections. Therefore, other laboratory techniques are required for detecting GBS more accurately. The proposed method "MALDI detection with analysis of peaks of TOF (MDAPT)" detects GBS directly from cultured broth with high sensitivity. Therefore, it can be an alternative method for GBS screening in pregnant women, thereby contributing to the prevention of severe GBS infectious diseases in neonates.

Identification and antimicrobial susceptibility profiles of Nocardia species clinically isolated in Japan.

The aims of the present study were to profile the antimicrobial susceptibility patterns of a diverse range of Nocardia species isolated in Japan, and to determine the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for species/complex identification. Identification of 153 clinical isolates was performed by full-length 16S rRNA gene sequencing as a reference method to evaluate the usefulness of MALDI-TOF MS identification. Antimicrobial susceptibility testing (AST) for 14 antibiotics was performed using the broth microdilution method against 146 of the isolates. Among the total 153 clinical isolates, Nocardia farcinica complex (25%) was the most common species, followed by Nocardia cyriacigeorgica (18%), Nocardia brasiliensis (9%), Nocardia nova (8%), and Nocardia otitidiscaviarum (7%). Among 150 isolates identified to the species/complex level by 16S rRNA gene sequencing, MALDI-TOF MS with the use of a supplemental Nocardia library (JMLD library ver.ML01) correctly identified 97.3% (n = 146) to the species/complex level and 1.3% (n = 2) to the genus level. Among the 146 Nocardia isolates that underwent AST, the susceptibilities were 100% to linezolid, 96% to amikacin, 94% to trimethoprim-sulfamethoxazole, and 76% to imipenem. None of the trimethoprim-sulfamethoxazole-resistant isolates carried either plasmid-mediated sulfonamide-resistant genes (sul1, sul2) or trimethoprim-resistant genes (dfrA).

Directed Evolution of the Stringency of the LuxR Vibrio fischeri Quorum Sensor without OFF-State Selection.

Stringency (low leak) is one of the most important specifications required for genetic circuits and induction systems, but it is challenging to evolve without sacrificing the maximum output level. This problem also comes from the absence of truly tunable negative selection methods. This paper reports that stringently switching variants can sometimes emerge with surprising frequency upon mutations. We randomly mutated the previously generated leaky variants of LuxR, the quorum-sensing transcription activator from Vibrio fischeri, to restore the stringency. We found as much as 10-20% of the entire population exhibited significantly improved signal-to-noise ratios compared with their parents. This indicated that these mutants arose by the loss of folding capability by accumulating destabilizing mutations, not by introducing rare adaptive mutations, thereby becoming AHL-dependent folders. Only four rounds of mutagenesis and ON-state selection resulted in the domination of the entire population by the improved variants with low leak, without direct selection pressure for stringency. With this surprising frequency, conversion into the "ligand-addicted folders" should be one of the prevailing modes of evolving stringency both in the laboratory and in nature, and the workflow described here provides a rapid and versatile method of improving the signal-to-noise ratio of various genetic switches.

Improvement of the dP-nucleoside-mediated herpes simplex virus thymidine kinase negative-selection system by manipulating dP metabolism genes.

A variety of positive/negative selection systems have been exploited as genome engineering tools and screening platforms for genetic switches. While numerous positive-selection systems are available, only a handful of negative-selection systems are useful for such applications. We previously reported a powerful negative-selection system using herpes simplex virus thymidine kinase (HsvTK) and the mutagenic nucleoside analog 6-(ß-d-2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido [4,5-c][1,2] oxazin-7-one (dP). Upon addition of 1000 nM dP, cells expressing HsvTK quickly die, with unprecedented efficacy. However, this selection procedure elevates the spontaneous mutation rate of the host cells by 10-fold due to the mutagenic nature of dP. To decrease the operative concentration of dP required for negative selection, we systematically created the strains of Escherichia coli either by removing or overexpressing genes involved in DNA/RNA metabolism. We found that over-expression of NupC and NupG (nucleoside uptake-related inner membrane transporters), Tsx (outer membrane transporter), NdK (nucleotide kinase) sensitized E. coli cells to dP. Simultaneous overexpression of these three genes (ndk-nupC-tsx) significantly improved the dP-sensitivity of E. coli, lowering the necessary operative concentration of dP for negative selection by 10-fold. This enabled robust and selective elimination of strains harboring chromosomally-encoded hsvtk simply by adding as low as 100 nM dP, which causes only a modest increase in the spontaneous mutation frequency as compared to the cells without hsvtk.

Validation of MALDI-TOF MS devices in reanalysis of unidentified pathogenic bacteria detected in blood cultures.

In hospital microbial laboratories, morphological and biochemical analyses are performed to identify pathogenic microbes;however, these procedures lack rapidity and accuracy. Recently, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been clinically utilized, and is expected to enable rapid and accurate microbial identification. We aimed to validate two MALDI-TOF MS devices available in Japan: the VITEK-MS (BioMérieux) and the Microflex LT (Bruker Daltonics). Clinically isolated bacteria, 100 samples in all, detected in blood cultures but incompletely identified by conventional procedures, were reanalyzed using the two devices. The VITEK-MS and Microflex LT, respectively, identified 49% (49/100) and 80% (80/100) of the tested bacteria at the species level, as well as 96% (96/100) and 95% (95/100) at the genus level. Among those reidentified strains, 26% (26/100) at the species level and 88% (88/100) at the genus level were concordant with each other, though three strains were unmatched. Moreover, four bacterial strains were unable to be identified using the VITEK-MS, versus five using the Microflex LT. MALDI-TOF MS devices can provide more rapid and accurate bacterial identification than ever before;however, the characteristics of each system were slightly different;therefore, it is necessary to understand the difference in performance of MALDI-TOF MS models.

Construction of a pathway to C50-ε-carotene.

Substrate tolerance of bacterial cyclases has been demonstrated in various contexts, but little is known about that of plant cyclases. Here, we tested two plant ε-cyclases to convert C50-lycopene, which we previously established by rounds of directed evolution. Unlike bacterial ß-cyclases, two-end cyclase from lettuce exhibited complete specificity against this molecule, indicating that this enzyme has some mechanism that exerts size-specificity. Arabidopsis one-end cyclase At-y2 showed detectable activity to C50-lycopene. Interestingly, we found that it functions as a two-end cyclase in a C50 context. Based on this observation, a possible model for substrate discrimination of this enzyme is proposed.

Genetically engineered biosynthetic pathways for nonnatural C60 carotenoids using C5-elongases and C50-cyclases in Escherichia coli.

While the majority of the natural carotenoid pigments are based on 40-carbon (C40) skeleton, some carotenoids from bacteria have larger C50 skeleton, biosynthesized by attaching two isoprene units (C5) to both sides of the C40 carotenoid pigment lycopene. Subsequent cyclization reactions result in the production of C50 carotenoids with diverse and unique skeletal structures. To produce even larger nonnatural novel carotenoids with C50 + C5 + C5 = C60 skeletons, we systematically coexpressed natural C50 carotenoid biosynthetic enzymes (lycopene C5-elongases and C50-cyclases) from various bacterial sources together with the laboratory-engineered nonnatural C50-lycopene pathway in Escherichia coli. Among the tested enzymes, the elongases and cyclases from Micrococcus luteus exhibited significant activity toward C50-lycopene, and yielded the novel carotenoids C60-flavuxanthin and C60-sarcinaxanthin. Moreover, coexpression of M. luteus elongase with Corynebacterium cyclase resulted in the production of C60-sarcinaxanthin, C60-sarprenoxanthin, and C60-decaprenoxanthin.

Nonnatural biosynthetic pathway for 2-hydroxylated xanthophylls with C50-carotenoid backbone.

Carotenoids are structurally diverse pigments with various important biological functions. There has been a large interest in the search for novel carotenoid structures, since only a slight structural changes can result in a drastic difference in their biological functions. Carotenoid-modifying enzymes show remarkable substrate promiscuity, allowing rapid access to a vast set of novel carotenoids by combinatorial biosynthesis. We previously constructed a nonnatural carotenoid biosynthetic pathway in Escherichia coli that can produce C50 carotenoids having a longer chain than their natural C40 counterparts. In this study, a carotenoid 2,2'-hydroxylase (crtG) from Brevundimonas sp. SD212 was coexpressed together with our laboratory-engineered C50-zeaxanthin and C50-astaxanthin biosynthetic pathways. We identified six novel nonnatural C50-xanthophylls, namely, C50-nostoxanthin, C50-caloxanthin, C50-adonixanthin, C50-4-ketonostoxanthin, C50-2-hydroxyastaxanthin, and C50-2,2'-dihydroxyastaxanthin.

Construction of a Nonnatural C60 Carotenoid Biosynthetic Pathway.

Longer-chain carotenoids have interesting physiological and electronic/photonic properties due to their extensive polyene structures. Establishing nonnatural biosynthetic pathways for longer-chain carotenoids in engineerable microorganisms will provide a platform to diversify and explore the potential of these molecules. We have previously reported the biosynthesis of nonnatural C50 carotenoids by engineering a C30-carotenoid backbone synthase (CrtM) from Staphylococcus aureus. In the present work, we conducted a series of experiments to engineer C60 carotenoid pathways. Stepwise introduction of cavity-expanding mutations together with stabilizing mutations progressively shifted the product size specificity of CrtM toward efficient synthases for C60 carotenoids. By coexpressing these CrtM variants with hexaprenyl diphosphate synthase, we observed that C60-phytoene accumulated together with a small amount of C65-phytoene, which is the largest carotenoid biosynthesized to date. Although these carotenoids failed to serve as a substrate for carotene desaturases, the C25-half of the C55-phytoene was accepted by the variant of phytoene desaturase CrtI, leading to accumulation of the largest carotenoid-based pigments. Continuing effort should further expand the scope of carotenoids, which are promising components for various biological (light-harvesting, antioxidant, and communicating) and nonbiological (photovoltaic, photonic, and field-effect transistor) systems.