A bifunctional methyltransferase in biosynthesis of antitumor antibiotic streptonigrin.

Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid antibiotic with broad and potent antitumor activity. STN structurally contains four methyl groups belonging to two types: C-methyl group and O-methyl groups. Here, we report the biochemical characterization of the O-methyltransferase StnQ2 that can catalyze both the methylation of a hydroxyl group and a carboxyl group in the biosynthesis of streptonigrin. This work not only provides a new insight into methyltransferases, but also advances the elucidation of the complete biosynthetic pathway of streptonigrin.

Cytochrome P450 Mining for Bufadienolide Diversification.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

Analysis of six cancer patients with persistent left superior vena cava identified during central venous access device placement via an intracavitary electrocardiogram.

Persistent left superior vena cava (PLSVC) is a rare congenital anomaly. PLSVC can be associated with clinically significant atrial septal defect (ASD) or ventricular septal defect (VSD). It is usually asymptomatic and accidentally detected during invasive procedures or imaging examinations. However, whether central venous access device (CVAD) can be placed and used in patients with PLSVC is controversial. A total of six patients were diagnosed with PLSVC and confirmed by chest CT among 3391 cancer patients who underwent CVAD placement via intracavitary electrocardiogram (IC-EKG) at the Venous Access Center (VAC) from May 2019 to December 2020. The CVADs (peripherally inserted central catheter in four patients and Ports in two patients) of these six patients were left in PLSVC. We analyzed changes in the P-wave in the IC-EKG during CVAD placement and the characteristics of the body surface electrocardiogram in these patients and discussed the catheter tip position in PLSVC. All six patients showed negative P-waves in lead II via IC-EKG from the beginning of catheterization: four patients showed negative P-waves and two showed biphasic P-waves in the body surface electrocardiogram (lead III) before catheterization. CVAD function was normal and no obvious complications were observed during the treatment of these patients. The total retention time of CVADs was 1537 days. For patients with a negative P-wave in lead II via IC-EKG during catheterization, especially in those with a negative or biphasic P-wave in lead III of the body surface electrocardiogram, PLSVC should be considered. CVAD insertion in patients with type I PLSVC is safe under certain conditions, with the proper tip position in the middle to lower part of PLSVC.

Flavoprotein StnP2 Catalyzes the ß-Carboline Formation during the Streptonigrin Biosynthesis.

ß-Carboline (ßC) alkaloids constitute a large family of indole alkaloids that exhibit diverse pharmacological properties, such as antitumor, antiviral, antiparasitic, and antimicrobial activities. Here, we report that a flavoprotein StnP2 catalyzes the dehydrogenation at C1-N2 of a tetrahydro-ß-carboline (THßC) generating a 3,4-dihydro-ß-carboline (DHßC), and the DHßC subsequently undergoes a spontaneous dehydrogenation to ßC formation involved in the biosynthesis of the antitumor agent streptonigrin. Biochemical characterization showed that StnP2 catalyzed the highly regio- and stereo-selective dehydrogenation, and StnP2 exhibits promiscuity toward different THßCs. This study provides an alternative kind of enzyme catalyzing the biosynthesis of ßC alkaloids and enhances the importance of flavoproteins.

A novel streptonigrin type alkaloid from the Streptomyces flocculus CGMCC 4.1223 mutant ΔstnA/Q2.

Streptonigrin (STN) is a highly functionalized aminoquinone alkaloid with broad and potent antitumor activities. Previously, the biosynthetic gene cluster of STN was identified in Streptomyces flocculus CGMCC 4.1223, revealing an α/ß-hydrolase (StnA) and a methyltransferase (StnQ2). In this work, a double mutant ΔstnA/Q2 was constructed by genetic manipulation and produced a novel derivative of STN, named as streptonigramide. Structure of streptonigramide was established by spectroscopic analyses. Its biosynthetic pathway has been proposed as well.

A comparative study on the tip position of peripherally inserted central catheter inserted at two forward P-wave amplitudes by intracavitary electrocardiogram in cancer patients.

BACKGROUND: This study aimed to compare the tip location of peripherally inserted central catheter (PICC) under two forward P-wave amplitudes (P-wave amplitude is the autonomous peak or P-wave amplitude is 50-80% of the QRS main wave) by intracavitary electrocardiogram (IC-EKG) to determine the PICC tip in optimal location thus avoiding catheter-related complications. METHODS: The data of 300 cancer patients with PICC insertion were collected retrospectively. For the observation group, the position of the catheter tip was left at the level when P wave amplitude was its autonomous peak (168 patients catheterized in 2018). While for the control group, the catheter tip was left at the level when the P wave amplitude was 50-80% of the QRS main wave (132 patients catheterized in 2017). Both groups of patients underwent the chest X-ray examination (CXR) after catheterization. The total compliance rate [PICC tip was located in the lower third of the Superior Vena Cava (SVC) and the Cavo-Atrial Junction (CAJ)], the optimal position compliance rate (PICC tip was located in the CAJ), and the incidence of the catheter tip malposition were compared between the two groups. The complications after catheterization including arrhythmia after catheterization within 24 hours, catheter-related thrombosis, catheter dysfunction, and catheter infection within 90 days were also compared. RESULTS: There was no difference in the total compliance rate of PICC tip position and the incidence of the catheter malposition in the two groups (P>0.05). But the optimal position compliance rate of the observation group was higher than that of the control group (P<0.05). There was no difference in the incidence of arrhythmia after catheterization within 24 hours of the two groups (P>0.05). The incidence of catheter-related thrombosis, catheter dysfunction, and catheter infection within 90 days in the observation group was lower than those in the control group (P<0.05). CONCLUSIONS: The PICC tip position at the autonomous peak of the P wave is significantly better than that at the P wave amplitude being 50-80% of the QRS main wave under the IC-EKG guidance for PICC insertion.

Association between ABO blood group and venous thrombosis related to the peripherally inserted central catheters in cancer patients.

OBJECTIVE: To evaluate the potential relation between the ABO blood group and the risk of venous thrombosis in cancer patients with peripherally inserted central catheters (PICCs). METHODS: The patients who underwent PICC catheterization in Beijing Cancer Hospital from January 2018 to October 2019 were retrospectively analyzed. The general information, disease diagnosis, catheterization situation, and complications were recorded for each patient. Further, the blood group status was identified using the hospital information systems. Logistic and Cox proportional hazard regression analyses were performed to identify the risk factors for symptomatic PICC-related thrombosis. RESULTS: Among the 2315 patients, 131 had symptomatic thrombosis after PICC catheterization. The incidence of symptomatic thrombosis was lower in patients with blood type O when compared with that in patients with blood types other than O. The history of venous thrombosis, tumor category, arm circumference, and insertion attempts are risk factors associated with the PICC-related venous thromboembolism (VTE). After multivariable adjustment, insertion attempts and the non-O blood type were observed to remain associated with thrombosis. CONCLUSION: The risk of PICC-related thrombosis in patients with non-O blood type is significantly higher than that in patients with blood type O.

The Influence of Venous Characteristics on Peripherally Inserted Central Catheter-Related Symptomatic Venous Thrombosis in Cancer Patients.

BACKGROUND: With increasing use, peripherally inserted central catheters (PICCs) are associated with the risk of venous thrombosis. Few studies have focused on the relationships between venous thrombosis and venous characteristics. This study aimed to identify effects of venous characteristics on symptomatic PICC-related venous thrombosis in cancer patients and explore the relationship between venous characteristics and blood flow velocity. METHODS: The data of patients who underwent placement of PICC were retrospectively studied between January 2015 and September 2017. Symptomatic PICC-related venous thrombosis was confirmed by ultrasound. Univariable, multivariable logistic regression analyses were performed to identify the risk factors associated with PICC-related venous thrombosis. In October 2017, 169 patients with PICCs were enrolled prospectively, and the relationships between blood flow velocity and venous characteristics were recorded and analyzed. RESULTS: A total of 2933 cancer patients were enrolled in this study; of these patients, 68 experienced symptomatic venous thrombosis. In the bivariate analysis, body mass index (BMI), history of venous thrombosis, triglycerides, tumor category, vessel diameter, vessel depth and arm circumference were associated with thrombosis. The multivariable analyses showed that arm circumference, vascular diameter, triglyceride level and tumor category were independent risk factors for thrombosis. Blood flow velocity was positively correlated with vessel depth and arm circumference but not with vessel diameter. CONCLUSION: Different venous characteristics can lead to different blood flow rates, which can affect the incidence of thrombosis. A vein depth of greater than 1.07cm or less than 0.57cm was associated with a higher incidence of PICC-related venous thrombosis, and the greater the arm circumference and vessel diameter, the greater the risk of venous thrombosis.

Naphthoquinone-Based Meroterpenoids from Marine-Derived Streptomyces sp. B9173.

Naphthoquinone-based meroterpenoids are hybrid polyketide-terpenoid natural products with chemical diversity and a broad range of biological activities. Here, we report the isolation of a group of naphthoquinone-containing compounds from Streptomyces sp. B9173, and their structures were elucidated by using a combination of spectroscopic techniques, including 1D, 2D NMR, and high-resolution mass (HRMS) analysis. Seven flaviogeranin congeners or intermediates, three of which were new, have been derived from common naphthoquinone backbone and subsequent oxidation, methylation, prenylation, and amino group incorporation. Both flaviogeranin B1 (1) and B (2) contain an amino group which was incorporated into the C8 of 1,3,6,8-terhydroxynaphthalene (THN). Flaviogeranin D (3) contains an intact C-geranylgeranyl residue attached to the C2 of THN, while the O-geranylgeranyl group of 2 links with the hydroxyl on the C2 site of THN. Four compounds were selected and tested for antibacterial activity and cytotoxicity, with 3 and flaviogeranin C2 (5) displaying potent activity against selected bacteria and cancer cell lines. In light of the structure features of isolated compounds and the biosynthetic genes, a biosynthetic pathway of naphthoquinone-based flaviogeranins has been proposed. These isolated compounds not only extend the structural diversity but also represent new insights into the biosynthesis of naphthoquinone-based meroterpenoids.

StnK2 catalysing a Pictet-Spengler reaction involved in the biosynthesis of the antitumor reagent streptonigrin.

Streptonigrin (STN, 1) is a highly functionalized aminoquinone alkaloid antibiotic with broad and potent antitumor activity. Previous isotope-labelling and genetic studies suggested that a ß-carboline alkaloid should be a key intermediate of STN biosynthesis and formed via a Pictet-Spengler (PS) reaction. Herein, StnK2 was biochemically characterized to be a Pictet-Spenglerase (PSase) catalysing the formation of a tetrahydro-ß-carboline (TH-ßC) scaffold from (2S,3S)-ß-methyl tryptophan and d-erythrose-4-phosphate. StnK2 can tolerate the alteration of tryptophan but only accept d-erythrose-4-phosphate as the aldehyde substrate, and StnK2 was identified to be R-specific for the newly formed chiral center. This work increases the diversities of Pictet-Spenglerase in nature and set a stage for the generation of streptonigrin derivatives by precursor-directed pathway engineering based on the flexible substrate selectivity of StnK2.

Xantholipin B produced by the stnR inactivation mutant Streptomyces flocculus CGMCC 4.1223 WJN-1.

Xantholipin is a polycyclic xanthone antibiotic that exhibits potent cytotoxic and antibacterial activity. In this study, a new xanthone-type antibiotic, xantholipin B (1), was isolated for the first time along with its known derivative, xantholipin (2), from strain WJN-1, an aminotransferase inactivation mutant of the streptonigrin-producer Streptomyces flocculus CGMCC 4.1223. The structure of 1 was established based on spectroscopic analysis and supports the previously proposed biosynthetic pathway as a key intermediate of 2. Moreover, 1 showed 3- to 10-fold greater cytotoxicity than 2 against a select panel of human cancer cell lines. In addition, 1 demonstrated powerful antimicrobial activity against both Gram-positive bacteria and fungi. Importantly, both 1 and 2 inhibited the methicillin-resistant strain Staphylococcus aureus Mu50, with the MIC value of 0.025 µg ml-1. The new structural features of 1 enrich the structural diversity of xantholipin family compounds and shed new light on the structure-activity relationship of 1 as a promising antitumor drug candidate.

New isocoumarins and alkaloid from Chinese insect medicine, Eupolyphaga sinensis Walker.

Two new isocoumarins (1 and 2), a new alkaloid (3), and a known N-acetyldopamine dimer (4) were isolated from the ethyl acetate extract of Chinese insect medicine Eupolyphaga sinensis. Their structures were elucidated on the basis of detailed spectroscopic investigations, such as 1D- and 2D NMR spectroscopy, as well as by means of HR-MS. The structure of 1 was firmly confirmed by X-ray crystallography, and the absolute configuration was revealed by experimental and computational optical rotation analyses. Cytotoxicities of 1-4 were measured in vitro against 10 selected cancer cell lines.

The biosynthetic products of Chinese insect medicine, Aspongopus chinensis.

A new oxazole (1) was obtained from Chinese insect medicine Aspongopus chinensis, along with three known N-acetyldopamine derivatives (2-4). Their structures were determined on the basis of NMR and ESI-MS analyses. The possible biosynthetic pathways of the isolated compounds are discussed. Cytotoxicities of those compounds against 10 selected cancer cells were measured in vitro.

Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum and other related genomes as revealed by LMW-GS genes at Glu-3 loci.

Phylogenetic relationships between the C, U, N, and M genomes of Aegilops species and the genomes of common wheat and other related species were investigated by using three types of low-molecular-weight glutenin subunit (LMW-GS) genes at Glu-3 loci. A total of 20 LMW-GS genes from Aegilops and Triticum species were isolated, including 11 LMW-m type and 9 LMW-i type genes. Particularly, four LMW-m type and three LMW-i type subunits encoded by the genes on the C, N, and U genomes possessed an extra cysteine residue at conserved positions, which could provide useful information for understanding phylogenetic relationships among Aegilops and Triticum genomes. Phylogenetic trees constructed by using either LMW-i or the combination of LMW-m and LMW-s, as well as analysis of all the three types of LMW-GS genes together, demonstrated that the C and U genomes were closely related to the A genome, whereas the N and M genomes were closely related to the D genome. Our results support previous findings that the A genome was derived from Triticum uratu, the B genome was from Aegilops speltoides, and the D genome was from Aegilops tauschii. In addition, phylogenetic relationships among different genomes analysed in this study support the concept that Aegilops is not monophyletic.