The Post-Polyketide Synthase Modification Mechanism in Hitachimycin Biosynthesis.

Hitachimycin is a bicyclic macrolactam antibiotic with (S)-ß-phenylalanine (ß-Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize ß-amino acids, modify the aminoacyl groups, and transfer the resultant dipeptide groups to the acyl carrier protein domains of polyketide synthases (PKSs) have been studied extensively, the post-PKS modification mechanism responsible for constructing the unique bicyclic structure of hitachimycin remains elusive. In this study, we first inactivated six genes encoding putative post-PKS modification enzymes, namely hitM1 to hitM6, in Streptomyces scabrisporus to determine their involvement in hitachimycin biosynthesis. The ΔhitM4 strain accumulated an all-trans-2,4,6,8,18-pentaene macrolactam, which was confirmed as a true intermediate in hitachimycin biosynthesis by cellular feeding experiments, and appears to be the initial intermediate in the post-PKS modification pathway. The ΔhitM1 strain accumulated 10-O-demethyl-10-oxohitachimycin (M1-A). In enzymatic experiments, M1-A was reduced by the NAD(P)H-dependent reductase HitM1 in the presence of NADPH. The product of the reaction catalyzed by HitM1 was converted to hitachimycin by the methyltransferase HitM6. We thus propose a plausible post-PKS modification mechanism for the biosynthesis of hitachimycin.

Prognostic impact of radiological tumor burden in patients with metastatic urothelial carcinoma treated with pembrolizumab.

PURPOSE: Radiological tumor burden has been reported to be prognostic in many malignancies in the immunotherapy era, yet whether it is prognostic in patients with metastatic urothelial carcinoma (mUC) treated with pembrolizumab remains uninvestigated. We sought to assess the predictive and prognostic value of radiological tumor burden in patients with mUC. METHODS: We performed a retrospective analysis of 308 patients with mUC treated with pembrolizumab. Radiological tumor burden was represented by baseline tumor size (BTS) and baseline tumor number (BTN). Optimal cut-off value of BTS was determined as 50 mm using the Youden index (small BTS: n = 194, large BTS: n = 114). Overall (OS), cancer-specific (CSS), progression-free survival (PFS), and objective response rate (ORR) were compared. Non-linear associations between BTS and OS and CSS were evaluated using restricted cubic splines. RESULTS: Patients with large BTS were less likely to have undergone the surgical resection of the primary tumor (P = 0.01), and more likely to have liver metastasis (P < 0.001) and more metastatic lesions (P < 0.001). On multivariable analyses controlling for the effects of confounders (resection of primary tumor, metastatic site, number of metastases and lactate dehydrogenase level), large BTS and high BTN were independently associated with worse OS (HR 1.52; P = 0.015, and HR 1.69; P = 0.018, respectively) and CSS (HR 1.59; P = 0.01, and HR 1.66; P = 0.031, respectively), but not PFS. Restricted cubic splines revealed BTS was correlated with OS and CSS in linear relationships. Additionally, large BTS was significantly predictive of lower ORR and complete response rate on univariable analyses (P = 0.041 and P = 0.032, respectively), but its association disappeared on multivariable analyses. CONCLUSION: Radiological tumor burden has independent prognostic value with a linear relationship in pembrolizumab-treated patients with mUC and might help drive the earlier introduction of second-line pembrolizumab and/or switching to subsequent therapies.

A case of mucosal-associated lymphoid tissue lymphoma of the urachus.

Introduction: Urachus carcinoma is a rare malignancy with an aggressive potential and a poor prognosis, and evidence is limited for its diagnosis and treatment. Case presentation: A 75-year-old man underwent fluorodeoxyglucose positron emission tomography/computed tomography for staging prostate cancer, and a mass (standardized uptake value max 9.5) was observed on the outside of the urinary bladder dome. T2-weighted magnetic resonance imaging showed the urachus and a low-intensity tumor, which suggested a malignant tumor. We suspected urachal carcinoma and performed total resection of the urachus and partial cystectomy. Pathological examination revealed mucosa-associated lymphoid tissue lymphoma with cells positive for CD20 and negative for CD3, CD5, and cyclin D1. After the surgery, no recurrence has been observed for more than 2 years. Conclusion: We encountered an extremely rare case of mucosa-associated lymphoid tissue lymphoma of the urachus. Surgical resection of the tumor provided an accurate diagnosis and good disease control.

Acyltransferase Domain Exchange between Two Independent Type†I Polyketide Synthases in the Same Producer Strain of Macrolide Antibiotics.

Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activity. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without any supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon-elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.

Molecular-targeted Therapy for Metastatic Renal Cell Carcinoma As First-line Therapy: A Single Institution 13-year Experience.

We aimed to identify the role of first-line monotherapy with vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKI) in patients with metastatic RCC. Eligible patients were categorized into three groups (favorable, intermediate, and poor risk) according to the International Metastatic RCC Database Consortium risk criteria. Overall survival (OS) was the primary endpoint. Survival was compared using the log-rank test. A total of 108 patients were retrospectively analyzed. The numbers of patients in the favorable-, intermediate-, and poor-risk groups were 32 (30%), 66 (61%), and 10 (9%), repestively. The median OS values in the entire cohort was 36 months (95% confidence interval [CI] 29-53). The median OS in the favorable, intermediate, and poor risk groups were 94 months (95% CI: 43-Not reached), 30 months (95% CI: 20-38), and 8 months (95% CI: 0-Not reached), respectively (p<0.05). Prior nephrectomy, clear cell histology, clinical T stage ≤2, no metastasis at the time of diagnosis, nivolumab beyond first-line therapy, and objective response to VEGFR-TKIs were factors significantly prolonging OS on univariate analysis. VEGFR-TKI monotherapy as first-line therapy was an effective treatment option for patients with metastatic clear cell RCC with favorable risk.

Structural optimization and structure-functional selectivity relationship studies of G protein-biased EP2 receptor agonists.

The modification of the novel G protein-biased EP2 agonist 1 has been investigated to improve its G protein activity and develop a better understanding of its structure-functional selectivity relationship (SFSR). The optimization of the substituents on the phenyl ring of 1, followed by the inversion of the hydroxyl group on the cyclopentane moiety led to compound 9, which showed a 100-fold increase in its G protein activity compared with 1 without any increase in ß-arrestin recruitment. Furthermore, SFSR studies revealed that the combination of meta and para substituents on the phenyl moiety was crucial to the functional selectivity.

Discovery of G Protein-Biased EP2 Receptor Agonists.

To identify G protein-biased and highly subtype-selective EP2 receptor agonists, a series of bicyclic prostaglandin analogues were designed and synthesized. Structural hybridization of EP2/4 dual agonist 5 and prostacyclin analogue 6, followed by simplification of the ω chain enabled us to discover novel EP2 agonists with a unique prostacyclin-like scaffold. Further optimization of the ω chain was performed to improve EP2 agonist activity and subtype selectivity. Phenoxy derivative 18a showed potent agonist activity and excellent subtype selectivity. Furthermore, a series of compounds were identified as G protein-biased EP2 receptor agonists. These are the first examples of biased ligands of prostanoid receptors.

Abnormal pairing of X and Y sex chromosomes during meiosis I in interspecific hybrids of Phodopus campbelli and P. sungorus.

Hybrid sterility plays an important role in the maintenance of species identity and promotion of speciation. Male interspecific hybrids from crosses between Campbell's dwarf hamster (Phodopus campbelli) and the Djungarian hamster (P. sungorus) exhibit sterility with abnormal spermatogenesis. However, the meiotic phenotype of these hybrids has not been well described. In the present work, we observed the accumulation of spermatocytes and apoptosis of spermatocyte-like cells in the testes of hybrids between P. campbelli females and P. sungorus males. In hybrid spermatocytes, a high frequency of asynapsis of X and Y chromosomes during the pachytene-like stage and dissociation of these chromosomes during metaphase I (MI) was observed. No autosomal univalency was observed during pachytene-like and MI stages in the hybrids; however, a low frequency of synapsis between autosomes and X or Y chromosomes, interlocking and partial synapsis between autosomal pairs, and γ-H2AFX staining in autosomal chromatin was observed during the pachytene-like stage. Degenerated MI-like nuclei were frequently observed in the hybrids. Most of the spermatozoa in hybrid epididymides exhibited head malformation. These results indicate that the pairing of X and Y chromosomes is more adversely affected than that of autosomes in Phodopus hybrids.

Enhancing the effect of the nanofiber network structure on thermoresponsive wettability switching.

This letter reports the enhancing effects of a nanofiber network structure on stimuli-responsive wettability switching. Thermoresponsive coatings composed of nanofibers were prepared by electrospinning from thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAAm). The nanofiber coatings showed a large amplitude of thermoresponsive change in the wettability from hydrophilic to hydrophobic states compared to a smooth cast film. In particular, the combination of the surface chemistry and unique topology of the electrospun nanofiber coatings enables a transition from the Wenzel state to the metastable Cassie-Baxter state with an increase in temperature and consequently an enhanced amplitude of change in the water contact angles: the apparent contact angle differences between 25 and 50 °C are Δθ*(25-50 °C )= 108 and 10° for the nanofiber coatings with a diameter of 830 nm and a smooth cast film, respectively. The fabrication of the 3D nanofiber network structure by electrospinning from stimuli-responsive materials is a promising option for highly responsive surfaces in wettability.

Top-down process based on electrospinning, twisting, and heating for producing one-dimensional carbon nanotube assembly.

Multiwalled carbon nanotube (MWNT)/poly(vinyl butyral) (PVB) composite nanofibers were prepared by electrospinning, successive twisting and heat treatment. The MWNTs were highly oriented in an electrified thin jet during electrospinning. The heat treatment of the twisted electrospun nanofiber yarns produced the characteristics of the CNT in the composite nanofiber yarns and enhanced their electrical properties, mechanical properties, and thermal properties. The electrical conductivity of the heated yarn was significantly enhanced and showed the maximum value of 154 S cm(-1) for the yarn heated at 400 °C. It is an order of magnitude higher than other electrospun CNT composite materials. These results demonstrated that the novel top-down process based on electrospinning, twisting, and heat treatment provide a promising option for simple and large-scale manufacture of CNT assemblies.

Control over Internal Structure of Liquid Crystal Polymer Nanofibers by Electrospinning.

Liquid crystal polymer nanofibers with a diameter ranging from 0.13 to 4.71 µm were prepared by electrospinning from a main-chain liquid crystalline polyester, BB-5(3-Me). WAXD measurements showed that the formation and orientation of the ordered structure in the electrospun fibers were controlled by the fiber diameter formed during electrospinning. For BB-5(3-Me), the SmA structure with two layer spacings was formed in the fiber during the electrospinning. Under optimal spinning conditions, the SmA structure is highly oriented in the fiber. In addition, annealing transformed the metastable SmA structure in the BB-5(3-Me) fiber into stable SmCA one.

Nonlinear optical detection of proteins based on localized surface plasmons in surface immobilized gold nanospheres.

A new nonlinear optical method is presented to detect proteins binding to a gold surface without using fluorescent-dye labeling. After exposure of the protein-binding surface to a gold nanosphere solution, the nanospheres are immobilized above a gold surface with a nanogap supported by the protein. The gold nanospheres immobilized on the gold surface show strong localized surface plasmon (LSP) resonance, and the formation of this structure results in a marked increase in the optical second harmonic (SH) activity of the gold surface arising from a large enhancement of the electric field localized adjacent to the nanospheres on the LSP resonance. The SH image, therefore, gives a high contrast ratio, 7.0:1, of protein-binding spots to control spots. The contrast ratio is much greater than those obtained by linear reflectivity imaging.

Scanning electro-optic microscope based on surface plasmon resonance.

An optical scanning probe microscope, based on surface plasmon resonance (SPR) in the attenuated total reflection geometry, is shown to successfully image the polarization structures in self-assembled monolayers of hemicyanine adsorbed on a gold surface. Application of an ac field to the tip gives rise to the linear electro-optic effect in the monolayer just below the tip and the local change in the refractive index of the monolayer was detected via the ac component of the reflected light intensity, in which SPR-based detection intensifies the ac component. Polarization structures in a monolayer can be clearly imaged by this technique.

Multichannel biosensing platform of surface-immobilized gold nanospheres for linear and nonlinear optical imaging.

What we believe to be a new label-free multichannel biosensing platform is proposed. It is composed of surface-immobilized gold nanospheres (SIGNs) above a gold surface with a nanogap supported by a merocyanine self-assembled monolayer. The circular SIGN spots with a diameter of 120 microm were arrayed for multichannel biosensing on a glass slide. Two kinds of sensing methods were examined: One is a reflectivity measurement of a blue ray and the other is a second-harmonic generation measurement. It was found that the SIGN system can be used as a promising platform for multichannel biosensing in both sensing methods.

Second-harmonic spectroscopy of surface immobilized gold nanospheres above a gold surface supported by self-assembled monolayers.

We have investigated linear and nonlinear optical properties of surface immobilized gold nanospheres (SIGNs) above a gold surface with a gap distance of a few nanometers. The nanogap was supported by amine or merocyanine terminated self-assembled monolayers (SAMs) of alkanethiolates. A large second-harmonic generation (SHG) was observed from the SIGN systems at localized surface plasmon resonance condition. The maximum enhancement factor of SHG intensity was found to be 3 x 10(5) for the SIGN system of nanospheres 100 nm in diameter with a gap distance of 0.8 nm. The corresponding susceptibility was estimated to be chi((2))=750 pmV (1.8 x 10(-6) esu). In the SIGN system supported with the merocyanine terminated SAMs, the SHG response was also resonant to the merocyanine in the nanogap. It was found that the SHG response of the SIGN systems is strongly frequency dependent. This leads us to conclude that the large chi((2)) is caused by enhanced electric fields at the localized surface plasmon resonance condition and is not due to an increase of the surface susceptibility following from the presence of the gold nanospheres. The observed SHG was consistent with the theoretical calculations involving Fresnel correction factors, based on the quasistatic approximation.