Deletion of the accramycin pathway-specific regulatory gene <i>accJ</i> activates the production of unrelated polyketide metabolites

The manipulation of regulatory genes has been employed to awaken cryptic metabolites in Streptomyces. Of particular interest in recent years is the effect of disruption of a pathway-specific gene to other biosynthetic pathways. Herein, we report the inactivation of the accramycin pathway-specific regulatory gene, accJ in Streptomyces sp. MA37 resulted in the production of unrelated polyketide metabolites. Through detailed mass spectrometric and spectroscopic analyses, and comparison with literature data, their structures were deduced as 3-methoxy-2-methyl-4H-pyran-4-one (1), zanthopyranone (2), propioveratrone (3), and TW94a (4). To the best of our knowledge, this is the first report of the isolation of 1–3 from bacteria. Compounds 1, 2, and 4 showed weak to moderate activity against Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Propioveratrone (3) displayed better inhibitory activity (MIC = 6.3 μg/mL) than ampicillin against multi-drug resistant E. faecium K60–39 clinical isolate (MIC = 25 μg/mL), suggesting a promising structural template for the drug development targeting Enterococcus isolates.</p

Fluorine Speciation Analysis Using Reverse Phase Liquid Chromatography Coupled Off-Line to Continuum Source Molecular Absorption Spectrometry (CS-MAS): Identification and Quantification of Novel Fluorinated Organic Compounds in Environmental and Biological Samples

Driven by increasing demand for the monitoring of industrial perfluorinated compounds (PFCs), the identification of novel fluorine containing compounds (FOCs) and the tracking of organofluorine drugs and their degradation products, there is a clear need for sensitive, fluorine-specific detection of unknown FOCs. Here we report the first ever direct fluorine-specific (speciation) method; capable of individually detecting untargeted FOCs in environmental and biological samples through the application of continuum source molecular absorption spectrometry (CS-MAS) using a commercial CS-AAS. Two model FOCs (2,4,6, trifluorobenzoic acid (TFBA) and 5-fluoroindol-5-carboxylic acid (FICA)) were used, achieving fluorine-specific detection across a range of 0.1 to 300 ng/mL fluorine, corresponding to a limit of detection of 4 pg F and 5.26 nM for both compounds. Both TFBA and FICA showed a similar response to CS-MAS detection, potentially enabling the quantification of fluorine content in novel FOCs without having molecular standards available. This paper also reports the use of reverse-phase high performance liquid chromatography (RP-HPLC) coupled off-line with CS-MAS for the identification of single organofluorines in a mixture of FOCs via fraction collection. The linear range of both FOCs was determined to be from 1 to 500 ng/mL. The limits of detection of those species were just above 1 ng/mL (100 pg) and can therefore compete with targeted analytical methods such as ESI-MS. Finally, as a proof of principle the analysis of a fluoride-containing groundwater sample from Ghana demonstrated that this method can be used in the detection of novel FOCs, with identification achieved through parallel ESI-MS. Coupled HPLC–CS-MAS/ESI-MS is the first analytical methodology capable of selectively detecting and identifying novel FOCs, making possible the quantification of all fluorine containing compounds in one sample. This is the necessary analytical requirement to perform <i>fluoronomics</i>

Long-Term Outcomes of Radio-Frequency Catheter Ablation on Ventricular Tachycardias Due to Arrhythmogenic Right Ventricular Cardiomyopathy: A Single Center Experience

<div><p>Aims</p><p>To summarize our experience of radiofrequency catheter ablation (RFCA) for recurrent drug-refractory ventricular tachycardias (VTs) due to arrhythmogenic right ventricular cardiomyopathy (ARVC) in our center over the past 11 years and its related factors.</p><p>Methods and Results</p><p>We reviewed 48 adults (mean age 39.9 ± 12.9 years, range: 14 to 65) who met the present ARVC diagnostic criteria and accepted RFCA for VTs from December 2004 to April 2016. The patients received a total of 70 procedures using two ablation approaches, the endocardial approach in 52 RFCAs, and the combined epicardial and endocardial approach (the combined approach) in 18 RFCAs. Kaplan-Meier survival analysis showed that the combined approach achieved better acute procedural success (<i>p</i> = 0.003) and better long-term outcomes (<i>p</i> = 0.028) than the endocardial approach. Patients who obtained acute procedural success with non-inducibility had better long-term outcomes (<i>p</i> < 0.001). COX regression of multivariate analysis showed that procedural success was the only factor that benefited long-term outcome, irrespective of the endocardial or the combined approach (<i>p</i> = 0.001). The rate of sudden cardiac death (SCD) in patients without procedural success was significantly higher than that in patients with procedural success (<i>p</i> = 0.005). All patients without implantable cardioverter defibrillator (ICD) implantation who had successful final RFCA survived.</p><p>Conclusions</p><p>The combined approach resulted in better procedural success and long-term VT-free survival compared with the endocardial approach in ARVC patients with recurrent VTs. Acute procedural success with non-inducibility was strongly related to better long-term VT-free survival and reduced SCD, irrespective of whether this was achieved by the endocardial approach or the combined approach.</p></div

The overall cumulative VT-free survival curve of all 70 procedures.

<p>The overall cumulative VT-free survival curve of all 70 procedures.</p

Multivariate analysis of VT-free survival in all patients by COX regression analysis.

<p>Multivariate analysis of VT-free survival in all patients by COX regression analysis.</p

General demographic data and clinical status.

<p>General demographic data and clinical status.</p

Directed Accumulation of Anticancer Depsipeptides by Characterization of Neoantimycins Biosynthetic Pathway and an NADPH-Dependent Reductase

Neoantimycins (NATs) are members of antimycin-types of depsipeptides with outstanding anticancer activities. We isolated NAT-A (<b>1</b>) and -F (<b>2</b>) from the fermentation extract of <i>Streptomyces conglobatus</i>. The NAT biosynthetic gene cluster (<i>nat</i> BGC) was identified by genome sequencing and bioinformatics analysis. <i>nat</i> BGC includes two nonribosomal peptide synthetase (NRPS) and one polyketide synthase (PKS) gene, and a gene cassette (10 genes), of which the encoded enzymes share high homology to the ones responsible for 3-formamidosalicylate (3-FAS) biosynthesis in the antimycin biosynthetic pathway. Heterologous expression of the partial <i>nat</i> BGC without the 3-FAS gene cassette in the antimycin producer, <i>Streptomyces albus</i> J1074, results in the production of <b>1</b> and <b>2</b>, suggesting that the <i>nat</i> BGC indeed directs NATs biosynthesis. Targeted in-frame deletion of the reductase gene (<i>natE</i>) abolished the production of <b>1</b> and <b>2</b> but accumulated two NAT derivatives, the known NAT-H (<b>3</b>) and a new NAT-I (<b>4</b>). Biochemical verification demonstrated that the recombinant NatE indeed catalyzes an NADPH-dependent reaction of <b>3</b> or <b>4</b> to <b>1</b> or <b>2</b>, respectively. Compound <b>3</b> presented significantly stronger activities against eight cancer cell lines than the ones using cisplatin, the clinical chemotherapy medicine. In particular, <b>3</b> displayed 559- and 57-fold higher activity toward human melanoma and cervix epidermoid carcinoma cells, respectively, compared with cisplatin. The new derivative, <b>4</b>, was 1.5- to 10.9-fold more active than cisplatin toward five cancer cell lines. The evaluation of NATs biosynthesis depicted here will pave the way to generate new NAT derivatives through rational pathway engineering

This series of pictures show the electrophysiological characteristics in the RV epicardium of an ARVC patient.

<p>(A) A concealed entrainment within the isthmus of the reentrant circuit. The post-pacing interval (PPI) was 320 ms, almost equal to the VT cycle length (326 ms). The interval between the stimulation signal to onset of QRS complex (S-QRS) was 108 ms (108/320 = 0.34), indicating that the location was within the center of the isthmus. (B) The sequential activation of fractionated potentials. (C) VT stopped during ablation. (D) The three-dimensional activation map (Ensite Navx Velocity) of this patient’s RV epicardium which shows a reentry in the RV free wall. (E) Fluorescence of the mapping catheters. DD: St Jude Medical DuoDeca catheter.</p

Multivariate analysis of VT-free survival in the endocardial approach group by COX regression analysis.

<p>Multivariate analysis of VT-free survival in the endocardial approach group by COX regression analysis.</p

The substrate maps of the RV endocardium and epicardium of an ARVC patient (CARTO3 system).

<p>(A) The fractionated potentials are marked as blue dots within a low voltage zone and ablation energy was delivered within the low voltage zone with fractionated potentials in the RV endocardium. (B) This is the substrate map of the RV epicardium and ablation marks within the low voltage zone with fractionated potentials of the same patient.</p