Biosynthesis of Rhizocticins, Antifungal Phosphonate Oligopeptides Produced by Bacillus subtilis ATCC6633

SummaryRhizocticins are phosphonate oligopeptide antibiotics containing the C-terminal nonproteinogenic amino acid (Z)-l-2-amino-5-phosphono-3-pentenoic acid (APPA). Here we report the identification and characterization of the rhizocticin biosynthetic gene cluster (rhi) in Bacillus subtilis ATCC6633. Rhizocticin B was heterologously produced in the nonproducer strain Bacillus subtilis 168. A biosynthetic pathway is proposed on the basis of bioinformatics analysis of the rhi genes. One of the steps during the biosynthesis of APPA is an unusual aldol reaction between phosphonoacetaldehyde and oxaloacetate catalyzed by an aldolase homolog RhiG. Recombinant RhiG was prepared, and the product of an in vitro enzymatic conversion was characterized. Access to this intermediate allows for biochemical characterization of subsequent steps in the pathway

Molecular Cloning and Heterologous Expression of the Dehydrophos Biosynthetic Gene Cluster

SummaryDehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad-spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of S. lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments

Evolutionary radiation of lanthipeptides in marine cyanobacteria

Lanthipeptides are ribosomally derived peptide secondary metabolites that undergo extensive posttranslational modification. Prochlorosins are a group of lanthipeptides produced by certain strains of the ubiquitous marine picocyanobacteria Prochlorococcus and Synechococcus. Unlike other lanthipeptide-producing bacteria, picocyanobacteria use an unprecedented mechanism of substrate promiscuity for the production of numerous and diverse lanthipeptides using a single lanthionine synthetase. Through a cross-scale analysis of prochlorosin biosynthesis genes-from genomes to oceanic populations-we show that marine picocyanobacteria have the collective capacity to encode thousands of different cyclic peptides, few of which would display similar ring topologies. To understand how this extensive structural diversity arises, we used deep sequencing of wild populations to reveal genetic variation patterns in prochlorosin genes. We present evidence that structural variability among prochlorosins is the result of a diversifying selection process that favors large, rather than small, sequence changes in the precursor peptide genes. This mode of molecular evolution disregards any conservation of the ancestral structure and enables the emergence of extensively different cyclic peptides through short mutational paths based on indels. Contrary to its fast-evolving peptide substrates, the prochlorosin lanthionine synthetase evolves under a strong purifying selection, indicating that the diversification of prochlorosins is not constrained by commensurate changes in the biosynthetic enzyme. This evolutionary interplay between the prochlorosin peptide substrates and the lanthionine synthetase suggests that structure diversification, rather than structure refinement, is the driving force behind the creation of new prochlorosin structures and represents an intriguing mechanism by which natural product diversity arises. Keywords: lanthipeptides; prochlorosin; RiPPs; Prochlorococcus; SynechococcusGordon and Betty Moore Foundation (Grant GBMF495

Towards a developmental state? Provincial economic policy in South Africa

This paper explores the meaning of the developmental state for spatial economic policy in South Africa. Two main questions are addressed: do provincial governments have a role to play in promoting economic prosperity, and to what extent do current provincial policies possess the attributes of a developmental state? These attributes are defined as the ability to plan longer term, to focus key partners on a common agenda, and to mobilise state resources to build productive capabilities. The paper argues that the developmental state must harness the power of government at every level to ensure that each part of the country develops to its potential. However, current provincial capacity is uneven, and weakest where support is needed most. Many provinces seem to have partial strategies and lack the wherewithal for sustained implementation. Coordination across government appears to be poor. The paper concludes by suggesting ways provincial policies could be strengthened

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the \u3e20 distinct compound classes is also reviewed, and commonalities are discussed

Synthesis of methylphosphonic acid by marine microbes: a source for methane in the aerobic ocean

Relative to the atmosphere, much of the aerobic ocean is supersaturated with methane; however, the source of this important greenhouse gas remains enigmatic. Catabolism of methylphosphonic acid by phosphorus-starved marine microbes, with concomitant release of methane, has been suggested to explain this phenomenon, yet methylphosphonate is not a known natural product, nor has it been detected in natural systems. Further, its synthesis from known natural products would require unknown biochemistry. Here we show that the marine archaeon Nitrosopumilus maritimus encodes a pathway for methylphosphonate biosynthesis and that it produces cell-associated methylphosphonate esters. The abundance of a key gene in this pathway in metagenomic data sets suggests that methylphosphonate biosynthesis is relatively common in marine microbes, providing a plausible explanation for the methane paradox

⁸⁹Zr-pembrolizumab imaging as a non-invasive approach to assess clinical response to PD-1 blockade in cancer

Background: Programmed cell death protein 1 (PD-1) antibody treatment is standard of care for melanoma and non-small-cell lung cancer (NSCLC). Accurately predicting which patients will benefit is currently not possible. Tumor uptake and biodistribution of the PD-1 antibody might play a role. Therefore, we carried out a positron emission tomography (PET) imaging study with zirconium-89 ( 89Zr)-labeled pembrolizumab before PD-1 antibody treatment. Patients and methods: Patients with advanced or metastatic melanoma or NSCLC received 37 MBq (1 mCi) 89Zr-pembrolizumab (∼2.5 mg antibody) intravenously plus 2.5 or 7.5 mg unlabeled pembrolizumab. After that, up to three PET scans were carried out on days 2, 4, and 7. Next, PD-1 antibody treatment was initiated. 89Zr-pembrolizumab tumor uptake was calculated as maximum standardized uptake value (SUV max) and expressed as geometric mean. Normal organ uptake was calculated as SUV mean and expressed as a mean. Tumor response was assessed according to (i)RECIST v1.1. Results: Eighteen patients, 11 with melanoma and 7 with NSCLC, were included. The optimal dose was 5 mg pembrolizumab, and the optimal time point for PET scanning was day 7. The tumor SUV max did not differ between melanoma and NSCLC (4.9 and 6.5, P = 0.49). Tumor 89Zr-pembrolizumab uptake correlated with tumor response (P trend = 0.014) and progression-free (P = 0.0025) and overall survival (P = 0.026). 89Zr-pembrolizumab uptake at 5 mg was highest in the spleen with a mean SUV mean of 5.8 (standard deviation ±1.8). There was also 89Zr-pembrolizumab uptake in Waldeyer's ring, in normal lymph nodes, and at sites of inflammation. Conclusion: 89Zr-pembrolizumab uptake in tumor lesions correlated with treatment response and patient survival. 89Zr-pembrolizumab also showed uptake in lymphoid tissues and at sites of inflammation

Interleukin-2 PET imaging in patients with metastatic melanoma before and during immune checkpoint inhibitor therapy

PURPOSE: Immune checkpoint inhibitors can induce a T cell-mediated anti-tumor immune response in patients with melanoma. Visualizing T cell activity using positron emission tomography (PET) might allow early insight into treatment efficacy. Activated tumor-infiltrating T cells express the high-affinity interleukin-2 receptor (IL-2R). Therefore, we performed a pilot study, using fluorine-18-labeled IL-2 ([18F]FB-IL2 PET), to evaluate whether a treatment-induced immune response can be detected. METHODS: Patients with metastatic melanoma received ~ 200 MBq [18F]FB-IL2 intravenously, followed by a PET/CT scan before and during immune checkpoint inhibitor therapy. [18F]FB-IL2 uptake was measured as standardized uptake value in healthy tissues (SUVmean) and tumor lesions (SUVmax). Response to therapy was assessed using RECIST v1.1. Archival tumor tissues were used for immunohistochemical analyses of T cell infiltration. RESULTS: Baseline [18F]FB-IL2 PET scans were performed in 13 patients. SUVmean at baseline was highest in the kidneys (14.2, IQR: 11.6-18.0) and liver (10.6, IQR: 8.6-13.4). In lymphoid tissues, uptake was highest in spleen (10.9, IQR: 8.8-12.4) and bone marrow (2.5, IQR: 2.1-3.0). SUVmax in tumor lesions (n = 41) at baseline was 1.9 (IQR: 1.7-2.3). In 11 patients, serial imaging was performed, three at week 6, seven at week 2, and one at week 4. Median [18F]FB-IL2 tumor uptake decreased from 1.8 (IQR: 1.7-2.1) at baseline to 1.7 (IQR: 1.4-2.1) during treatment (p = 0.043). Changes in [18F]FB-IL2 tumor uptake did not correlate with response. IL-2R expression in four archival tumor tissues was low and did not correlate with baseline [18F]FB-IL2 uptake. No [18F]FB-IL2-related side effects occurred. CONCLUSION: PET imaging of the IL-2R, using [18F]FB-IL2, is safe and feasible. In this small patient group, serial [18F]FB-IL2-PET imaging did not detect a treatment-related immune response. TRIAL REGISTRATION: Clinicaltrials.gov : NCT02922283; EudraCT: 2014-003387.20

Setting things straight: a comparison of measures of saccade trajectory deviation

In eye movements, saccade trajectory deviation has often been used as a physiological operationalization of visual attention, distraction, or the visual system’s prioritization of different sources of information. However, there are many ways to measure saccade trajectories and to quantify their deviation. This may lead to noncomparable results and poses the problem of choosing a method that will maximize statistical power. Using data from existing studies and from our own experiments, we used principal components analysis to carry out a systematic quantification of the relationships among eight different measures of saccade trajectory deviation and their power to detect the effects of experimental manipulations, as measured by standardized effect size. We concluded that (1) the saccade deviation measure is a good default measure of saccade trajectory deviation, because it is somewhat correlated with all other measures and shows relatively high effect sizes for two well-known experimental effects; (2) more generally, measures made relative to the position of the saccade target are more powerful; and (3) measures of deviation based on the early part of the saccade are made more stable when they are based on data from an eyetracker with a high sampling rate. Our recommendations may be of use to future eye movement researchers seeking to optimize the designs of their studies