The Xenorhabdus nematophila LrhA transcriptional regulator modulates production of γ-keto-N-acyl amides with inhibitory activity against mutualistic host nematode egg hatching.

Xenorhabdus nematophila is a symbiotic Gammaproteobacterium that produces diverse natural products that facilitate mutualistic and pathogenic interactions in their nematode and insect hosts, respectively. The interplay between X. nematophila secondary metabolism and symbiosis stage is tuned by various global regulators. An example of such a regulator is the LysR-type protein transcription factor LrhA, which regulates amino acid metabolism and is necessary for virulence in insects and normal nematode progeny production. Here, we utilized comparative metabolomics and molecular networking to identify small molecule factors regulated by LrhA and characterized a rare γ-ketoacid (GKA) and two new N-acyl amides, GKA-Arg (1) and GKA-Pro (2) which harbor a γ-keto acyl appendage. A lrhA null mutant produced elevated levels of compound 1 and reduced levels of compound 2 relative to wild type. N-acyl amides 1 and 2 were shown to be selective agonists for the human G-protein-coupled receptors (GPCRs) C3AR1 and CHRM2, respectively. The CHRM2 agonist 2 deleteriously affected the hatch rate and length of Steinernema nematodes. This work further highlights the utility of exploiting regulators of host-bacteria interactions for the identification of the bioactive small molecule signals that they control. IMPORTANCE: Xenorhabdus bacteria are of interest due to their symbiotic relationship with Steinernema nematodes and their ability to produce a variety of natural bioactive compounds. Despite their importance, the regulatory hierarchy connecting specific natural products and their regulators is poorly understood. In this study, comparative metabolomic profiling was utilized to identify the secondary metabolites modulated by the X. nematophila global regulator LrhA. This analysis led to the discovery of three metabolites, including an N-acyl amide that inhibited the egg hatching rate and length of Steinernema carpocapsae nematodes. These findings support the notion that X. nematophila LrhA influences the symbiosis between X. nematophila and S. carpocapsae through N-acyl amide signaling. A deeper understanding of the regulatory hierarchy of these natural products could contribute to a better comprehension of the symbiotic relationship between X. nematophila and S. carpocapsae.

Metabolomics and Genomics Enable the Discovery of a New Class of Nonribosomal Peptidic Metallophores from a Marine Micromonospora.

Although microbial genomes harbor an abundance of biosynthetic gene clusters, there remain substantial technological gaps that impair the direct correlation of newly discovered gene clusters and their corresponding secondary metabolite products. As an example of one approach designed to minimize or bridge such gaps, we employed hierarchical clustering analysis and principal component analysis (hcapca, whose sole input is MS data) to prioritize 109 marine Micromonospora strains and ultimately identify novel strain WMMB482 as a candidate for in-depth "metabologenomics" analysis following its prioritization. Highlighting the power of current MS-based technologies, not only did hcapca enable the discovery of one new, nonribosomal peptide bearing an incredible diversity of unique functional groups, but metabolomics for WMMB482 unveiled 16 additional congeners via the application of Global Natural Product Social molecular networking (GNPS), herein named ecteinamines A-Q (1-17). The ecteinamines possess an unprecedented skeleton housing a host of uncommon functionalities including a menaquinone pathway-derived 2-naphthoate moiety, 4-methyloxazoline, the first example of a naturally occurring Ψ[CH2NH] "reduced amide", a methylsulfinyl moiety, and a d-cysteinyl residue that appears to derive from a unique noncanonical epimerase domain. Extensive in silico analysis of the ecteinamine (ect) biosynthetic gene cluster and stable isotope-feeding experiments helped illuminate the novel enzymology driving ecteinamine assembly as well the role of cluster collaborations or "duets" in producing such structurally complex agents. Finally, ecteinamines were found to bind nickel, cobalt, zinc, and copper, suggesting a possible biological role as broad-spectrum metallophores.

Colorectal cancer incidence trends by tumour location among adults of screening-age in England: a population-based study.

AIM: Proximal and distal colorectal cancers (CRCs) exhibit different clinical, molecular and biological patterns. The aim of this study was to determine temporal trends in the age-standardized incidence rates (ASIRs) of proximal and distal CRC following the introduction of the English Bowel Cancer Screening Programme (BCSP) in 2006. METHOD: The National Cancer Registration and Analysis Service database was used to identify incident cases of CRC among adults of screening age (60-74 years) between 2001 and 2017. ASIRs were calculated using the European Standard Population 2013 and incidence trends analysed by anatomical subsite (proximal, caecum to descending colon; distal, sigmoid to rectum), sex and Index of Multiple Deprivation (IMD) quintile using Joinpoint regression software. RESULTS: Between 2001 and 2017, 541 515 incident cases of CRC were diagnosed [236 167 proximal (43.6%) and 305 348 distal (56.4%)]. A marginal reduction in the proximal ASIR was noted from 2008 [annual percentage change (APC) -1.4% (95% CI -2.0% to -0.9%)] compared with a greater reduction in distal ASIR from 2011 to 2014 [APC -6.6% (95% CI -11.5% to -1.5%)] which plateaued thereafter [APC -0.5% (95% CI -3.2% to 2.2%)]. Incidence rates decreased more rapidly in men than women. Adults in IMD quintiles 4-5 experienced the greatest reduction in distal tumours [APC -3.5% (95% CI -4.3% to -2.7%)]. CONCLUSION: Following the introduction of the English BCSP, the incidence of CRC has subsequently reduced among adults of screening age, with this trend being most pronounced in distal tumours and in men. There is also evidence of a reduction in the deprivation gap for distal tumour incidence. Strategies to improve the detection of proximal tumours are warranted.

Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A.

Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) comprise biosynthetic pathways that provide access to diverse, often bioactive natural products. Metabolic engineering can improve production metrics to support characterization and drug-development studies, but often native hosts are difficult to genetically manipulate and/or culture. For this reason, heterologous expression is a common strategy for natural product discovery and characterization. Many bacteria have been developed to express heterologous biosynthetic gene clusters (BGCs) for producing polyketides and nonribosomal peptides. In this article, we describe tools for using Pseudomonas putida, a Gram-negative soil bacterium, as a heterologous host for producing natural products. Pseudomonads are known to produce many natural products, but P. putida production titers have been inconsistent in the literature and often low compared to other hosts. In recent years, synthetic biology tools for engineering P. putida have greatly improved, but their application towards production of natural products is limited. To demonstrate the potential of P. putida as a heterologous host, we introduced BGCs encoding the synthesis of prodigiosin and glidobactin A, two bioactive natural products synthesized from a combination of PKS and NRPS enzymology. Engineered strains exhibited robust production of both compounds after a single chromosomal integration of the corresponding BGC. Next, we took advantage of a set of genome-editing tools to increase titers by modifying transcription and translation of the BGCs and increasing the availability of auxiliary proteins required for PKS and NRPS activity. Lastly, we discovered genetic modifications to P. putida that affect natural product synthesis, including a strategy for removing a carbon sink that improves product titers. These efforts resulted in production strains capable of producing 1.1 g/L prodigiosin and 470 mg/L glidobactin A.

Bacillibactins E and F from a Marine Sponge-Associated Bacillus sp.

Chemical investigation of a marine sponge-associated Bacillus sp. led to the discovery of bacillibactins E and F (1 and 2). Despite containing the well-established cyclic triester core of iron-binding natural products such as enterobactin, bacillibactins E and F (1 and 2) are the first bacterial siderophores that contain nicotinic and benzoic acid moieties. The structures of the new compounds, including their absolute configurations, were determined by extensive spectroscopic analyses and Marfey's method. A plausible biosynthetic pathway to 1 and 2 is proposed; this route bears great similarity to other previously established bacillibactin-like pathways but appears to differentiate itself by a promiscuous DhbE, which likely installs the nicotinic moiety of 1 and the benzoic acid group of 2.

Outcomes following local excision of early anal squamous cell carcinomas of the anal canal and perianal margin.

AIM: There is a paucity of data on outcomes from local excision (LE) of early anal squamous cell carcinomas (ASCCs). This study aimed to assess survival outcomes according to tumour location, perianal (PAT) or anal canal (ACT), and to determine factors associated with R1 excision and outcomes according to T-category. METHODS: This was a retrospective cohort study of consecutive patients with early ASCC treated by LE from 2007 to 2019. Data were collected on baseline demographics, tumour location, staging, excision histology, adjuvant treatment, site and timing of recurrence. The main outcome measures were R1 resection, locoregional recurrence (LRR), disease-free survival and overall survival. RESULTS: Of 367 patients treated for ASCC, 39 (10.6%) patients with complete follow-up data underwent LE: 15 ACTs and 24 PATs. R1 resections were obtained in 27 patients (69.2%) and occurred more frequently in ACTs than PATs (93.3% vs. 54.2%, P = 0.006). Eighteen of 27 patients (66.7%) received adjuvant therapy (chemoradiotherapy [n = 11], radiotherapy alone [n = 7]) for R1 excision or re-excision, following which LRR developed in one of 10 (10.0%) patients in the ACT cohort and one of eight (12.5%) patients in the PAT cohort. There was no difference in 5-year LRR-free survival (82.0% vs. 70.1%, P = 0.252), disease-free survival (58.2% vs. 78.4%, P = 0.200) or overall survival (86.2% vs. 95.7%, P = 0.607) between the ACT and PAT cohorts. CONCLUSIONS: LE is a feasible treatment option for early ASCCs of the perianal margin but not the anal canal. Acceptable long-term outcomes can still be achieved with adjuvant therapy in the presence of a positive margin. Larger prospective studies to assess LE as a treatment strategy, such as the ACT3 trial, are warranted.

Optimisation of a key cross-coupling reaction towards the synthesis of a promising antileishmanial compound.

During the course of a research program aimed at identifying novel antileishmanial compounds, a multi-gram synthesis of N-(trans-4-((4-methoxy-3-((R)-3-methylmorpholino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)cyclohexyl)-2-methylpropane-1-sulfonamide (( R )-1) was required. This letter describes optimisation of the reaction conditions and protecting group strategy for a key Buchwald-Hartwig coupling, delivering the required quantities of ( R )-1, as well as further compounds in the series.

An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases in Myxococcus xanthus DK1622.

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs.IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs.

Alanine Scanning of YbdZ, an MbtH-like Protein, Reveals Essential Residues for Functional Interactions with Its Nonribosomal Peptide Synthetase Partner EntF.

Nonribosomal peptide synthetases (NRPSs) are megasynthetases that require complex and specific interactions between multiple domains and proteins to functionally produce a metabolite. MbtH-like proteins (MLPs) are integral components of many NRPSs and interact directly with the adenylation domain of the megasynthetases to stimulate functional enzymology. All of the MLP residues that are essential for functional interactions between the MLP and NRPS have yet to be defined. Here we probe the interactions between YbdZ, an MLP, and EntF, an NRPS, from Escherichia coli by performing a complete alanine scan of YbdZ. A phenotypic screen identified 11 YbdZ variants that are unable to replace the wild-type MLP, and these YbdZ variants were characterized using a series of in vivo and in vitro assays in an effort to explain why functional interactions with EntF were disrupted. All of the YbdZ variants enhanced the solubility of overproduced EntF, suggesting they were still capable of direct interactions with the megasynthase. Conversely, we show that EntF also influences the solubility of YbdZ and its variants. In vitro biochemical analyses of EntF function with each of the YbdZ variants found the impact that an amino acid substitution will have on NRPS function is difficult to predict, highlighting the complex interaction between these proteins.

Antitrypanosomal 8-Hydroxy-Naphthyridines Are Chelators of Divalent Transition Metals.

The lack of information regarding the mechanisms of action (MoA) or specific molecular targets of phenotypically active compounds can prove a barrier to their development as chemotherapeutic agents. Here, we report the results of our orthogonal genetic, molecular, and biochemical studies to determine the MoA of a novel 7-substituted 8-hydroxy-1,6-naphthyridine (8-HNT) series that displays promising activity against Trypanosoma brucei and Leishmania donovani High-throughput loss-of-function genetic screens in T. brucei highlighted two probable zinc transporters associated with resistance to these compounds. These transporters localized to the parasite Golgi apparatus. Directed by these findings, the role of zinc and other divalent cations in the MoA of these compounds was investigated. 8-HNT compounds were found to directly deplete intracellular levels of Zn2+, while the addition of exogenous Zn2+ and Fe2+ reduced the potency of compounds from this series. Detailed biochemical analyses confirmed that 8-HNT compounds bind directly to a number of divalent cations, predominantly Zn2+, Fe2+, and Cu2+, forming 2:1 complexes with one of these cations. Collectively, our studies demonstrate transition metal depletion, due to chelation, as the MoA of the 8-HNT series of compounds. Strategies to improve the selectivity of 8-HNT compounds are discussed.

2,4-Diamino-6-methylpyrimidines for the potential treatment of Chagas' disease.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, affects 8-10 million people across the Latin American population and is responsible for around 12,500 deaths per annum. The current frontline treatments, benznidazole and nifurtimox, are associated with side effects and lack efficacy in the chronic stage of the disease, leading to an urgent need for new treatments. A high throughput screening campaign against the physiologically relevant intracellular form of the parasite identified a series of 2,4-diamino-6-methylpyrimidines. Demonstrating the series did not work through the anti-target TcCYP51, and was generally cytocidal, confirmed its suitability for further development. This study reports the optimisation of selectivity and metabolic stability of the series and identification of a suitable lead for further optimisation.

Characterization of the Functional Variance in MbtH-like Protein Interactions with a Nonribosomal Peptide Synthetase.

Many nonribosomal peptide synthetases (NRPSs) require MbtH-like proteins (MLPs) for solubility or for activation of amino acid substrate by the adenylation domain. MLPs are capable of functional crosstalk with noncognate NRPSs at varying levels. Using enterobactin biosynthesis in Escherichia coli as a model MLP-dependent NRPS system, we use in vivo and in vitro techniques to characterize how seven noncognate MLPs influence the function of the enterobactin NRPS EntF when the cognate MLP, YbdZ, is absent. Using a series of in vitro assays to analyze EntF solubility, adenylation, aminoacylation, and in vitro enterobactin production, we show that interactions between MLPs and NRPSs are multifaceted and more complex than previously appreciated. We separate MLP influence on solubility and function in a manner that shows altered solubility is not indicative of a functional MLP/NRPS pair. Although much of the functional variation among these noncognates can be explained by differences in EntF affinity for an MLP or the extent an MLP alters EntF l-Ser affinity, we demonstrate that MLPs can have a broader impact beyond solubility and adenylation. First, we show that a noncognate MLP can affect formation of l-Ser-S-EntF. Second, under in vitro conditions saturating for substrate and MLP, enterobactin production remains compromised in the absence of an appropriate MLP partner. These data suggest that we expand our investigations into how the MLPs influence NRPS enzymology. A more detailed understanding of these influences will be essential for downstream engineering of hybrid NRPS systems.

Core Outcomes for Colorectal Cancer Surgery: A Consensus Study.

BACKGROUND: Colorectal cancer (CRC) is a major cause of worldwide morbidity and mortality. Surgical treatment is common, and there is a great need to improve the delivery of such care. The gold standard for evaluating surgery is within well-designed randomized controlled trials (RCTs); however, the impact of RCTs is diminished by a lack of coordinated outcome measurement and reporting. A solution to these issues is to develop an agreed standard "core" set of outcomes to be measured in all trials to facilitate cross-study comparisons, meta-analysis, and minimize outcome reporting bias. This study defines a core outcome set for CRC surgery. METHODS AND FINDINGS: The scope of this COS includes clinical effectiveness trials of surgical interventions for colorectal cancer. Excluded were nonsurgical oncological interventions. Potential outcomes of importance to patients and professionals were identified through systematic literature reviews and patient interviews. All outcomes were transcribed verbatim and categorized into domains by two independent researchers. This informed a questionnaire survey that asked stakeholders (patients and professionals) from United Kingdom CRC centers to rate the importance of each domain. Respondents were resurveyed following group feedback (Delphi methods). Outcomes rated as less important were discarded after each survey round according to predefined criteria, and remaining outcomes were considered at three consensus meetings; two involving international professionals and a separate one with patients. A modified nominal group technique was used to gain the final consensus. Data sources identified 1,216 outcomes of CRC surgery that informed a 91 domain questionnaire. First round questionnaires were returned from 63 out of 81 (78%) centers, including 90 professionals, and 97 out of 267 (35%) patients. Second round response rates were high for all stakeholders (>80%). Analysis of responses lead to 45 and 23 outcome domains being retained after the first and second surveys, respectively. Consensus meetings generated agreement on a 12 domain COS. This constituted five perioperative outcome domains (including anastomotic leak), four quality of life outcome domains (including fecal urgency and incontinence), and three oncological outcome domains (including long-term survival). CONCLUSION: This study used robust consensus methodology to develop a core outcome set for use in colorectal cancer surgical trials. It is now necessary to validate the use of this set in research practice.

A Systematic Review of Local Excision After Neoadjuvant Therapy for Rectal Cancer: Are ypT0 Tumors the Limit?

BACKGROUND: Neoadjuvant therapy reduces local recurrence after radical surgery for rectal cancer with complete pathological response in 15% to 25% of patients. Radical surgery is associated with significant morbidity that may be avoided by local excision in selected cases. OBJECTIVE: This systematic review aimed to determine the oncological outcomes and morbidity of local excision after neoadjuvant therapy. DATA SOURCES: Data sources included MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials databases. STUDY SELECTION: A systematic search of the databases using validated terms for rectal cancer, neoadjuvant therapy, and local excision was conducted. INTERVENTIONS: Neoadjuvant therapy and local excision were the included interventions. MAIN OUTCOME MEASURES: Pooled local recurrence, median survival, and pooled morbidity were measured. RESULTS: Twenty unique studies were included (14 cohort, 5 comparative cohort, and 1 randomized controlled trial), describing 1068 patients. Patient choice, prohibitive comorbidity, good clinical response, and early stage disease were the most frequent indications for local excision. Pretreatment T2 and T3 tumors accounted for 46.4% and 30.7% of cases. Long-course treatment was administered in all of the studies, except to a cohort of 64 patients who received short-course radiotherapy. Pooled complete clinical response was 45.8% (95% CI, 31.4%-60.5%), and pooled complete pathological response was 44.2% (95% CI, 36.4%-52.0%). Median follow-up was 54 months (range, 12-81 months). ypT0 tumors had a pooled local recurrence rate of 4.0% (95% CI, 1.9%-6.9%) and a median disease-free survival rate of 95.0% (95% CI, 87.4%-100%). Pooled local recurrence and median disease-free survival rates for ypT1 tumors or higher were 21.9% (95% CI, 15.9%-28.5%) and 68.0% (58.3%-69.0%). Pooled incidence of complications was 23.2% (95% CI, 15.7%-31.7%), with suture-line dehiscence reported in 9.9% (95% CI, 4.8%-16.7%). LIMITATIONS: Limitations included study quality, high risk of selection bias and detection bias in study designs, and limited sample sizes. CONCLUSIONS: Local excision after neoadjuvant therapy should only be considered a curative treatment if complete pathological response is obtained. Given the high rate of local recurrence among incomplete responders, future studies should focus on predicting patients who will achieve complete pathological response.

Structure of a cupin protein Plu4264 from Photorhabdus luminescens subsp. laumondii TTO1 at 1.35 Å resolution.

Proteins belonging to the cupin superfamily have a wide range of catalytic and noncatalytic functions. Cupin proteins commonly have the capacity to bind a metal ion with the metal frequently determining the function of the protein. We have been investigating the function of homologous cupin proteins that are conserved in more than 40 species of bacteria. To gain insights into the potential function of these proteins we have solved the structure of Plu4264 from Photorhabdus luminescens TTO1 at a resolution of 1.35 Å and identified manganese as the likely natural metal ligand of the protein.

The association between referral source and outcome in patients with colorectal cancer.

AIM: The colorectal two-week wait fast track (FT) referral system was nationally implemented in the UK in 2000 to ensure patients with colorectal cancer (CRC) received prompt access to specialized services. The aim of this study was to determine the association between the mechanism of referral to colorectal services and the 5-year outcomes for patients with CRC. METHODS: Consecutive patients with newly diagnosed CRC presenting between October 2002 and September 2004 were identified retrospectively. Analysis for survival and recurrence of disease at 5 years from presentation was undertaken. Outcomes for patients were compared between fast track (FT), non-fast track (NFT) and emergency referral (ER) routes, using Kaplan-Meier survival estimates. RESULTS: Out of 189 patients, 96 (51%) presented via the FT, 41 (22.5%) via the NFT and 52 (27.5%) via the ER referral route. The 5-year overall survival was 52.6% ± 5.1, 41.5% ± 7.7 and 38.5% ± 6.7 for the FT-, NFT- and ER groups respectively (p = 0.075). The 5-year cancer specific survival was 60.3% ± 5.2, 58.8% ± 5.3 and 43.5% ± 7.2 for the FT-, NFT- and ER groups respectively (p = 0.056). Patients referred as emergencies had worse 5-year overall survival; 49.3% ± 4.3 (FT&NFT) vs. 38.5% ± 6.7 (ER) (p = 0.042) and 5-year cancer specific survival 59.8% ± 4.4 (FT&NFT) vs. 43.5% ± 7.2 (ER) (p = 0.016). A total of 136 patients (FT n = 71, NFT n = 34, ER n = 31) underwent potentially curative surgery. Differences in 5-year survival did not reach statistical significance in these patients. CONCLUSION: Referral route to specialist services for patients with CRC via the fast track pathway compared to non-fast track pathway was not associated with improved survival.

Structure-Guided Design and Synthesis of a Pyridazinone Series of Trypanosoma cruzi Proteasome Inhibitors.

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.

Analyses of MbtB, MbtE, and MbtF suggest revisions to the mycobactin biosynthesis pathway in Mycobacterium tuberculosis.

The production of mycobactin (MBT) by Mycobacterium tuberculosis is essential for this bacterium to access iron when it is in an infected host. Due to this essential function, there is considerable interest in deciphering the mechanism of MBT assembly, with the goal of targeting select biosynthetic steps for antituberculosis drug development. The proposed scheme for MBT biosynthesis involves assembly of the MBT backbone by a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) megasynthase followed by the tailoring of this backbone by N(6) acylation of the central l-Lys residue and subsequent N(6)-hydroxylation of the central N(6)-acyl-l-Lys and the terminal caprolactam. A complete testing of this hypothesis has been hindered by the inability to heterologously produce soluble megasynthase components. Here we show that soluble forms of the NRPS components MbtB, MbtE, and MbtF are obtained when these enzymes are coproduced with MbtH. Using these soluble enzymes we determined the amino acid specificity of each adenylation (A) domain. These results suggest that the proposed tailoring enzymes are actually involved in precursor biosynthesis since the A domains of MbtE and MbtF are specific for N(6)-acyl-N(6)-hydroxy-l-Lys and N(6)-hydroxy-l-Lys, respectively. Furthermore, the preference of the A domain of MbtB for l-Thr over l-Ser suggests that the megasynthase produces MBT derivatives with ß-methyl oxazoline rings. Since the most prominent form of MBT produced by M. tuberculosis lacks this ß-methyl group, a mechanism for demethylation remains to be discovered. These results suggest revisions to the MBT biosynthesis pathway while also identifying new targets for antituberculosis drug development.

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression.

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

Demographic trends in the incidence of malignant appendiceal tumours in England between 1995 and 2016: Population-based analysis.

AIMS: Recent data suggest that the incidence of malignant appendiceal tumours is increasing. This study aimed to determine temporal trends in the incidence of malignant appendiceal tumours within England and a possible influence by demographic factors. METHODS: All incident cases of appendiceal tumours in patients aged 20 years and above were identified from the National Cancer Registration and Analysis Service database between 1995 and 2016 using ICD-9/10 codes. Cancers were categorized according to histology. Joinpoint regression analysis was used to investigate changes in age-standardized incidence rates by age, sex, histological subtype and index of multiple deprivation quintiles, based on socioeconomic domains (income, employment, education, health, crime, barriers to housing and services and living environment). Average annual per cent changes (AAPCs) were estimated by performing Monte-Carlo permutation analysis. RESULTS: A total of 7333 tumours were diagnosed and 7056 patients were analysed, comprising 3850 (54.6 per cent) neuroendocrine tumours (NETs), 1892 (26.8 per cent) mucinous adenocarcinomas and 1314 (18.6 per cent) adenocarcinoma (not otherwise specified). The overall incidence of appendiceal tumours increased from 0.3 per 100 000 to 1.6 per 100 000 over the study interval. Incidence rate increases of comparable magnitude were observed across all age groups, but the AAPC was highest among patients aged 20-29 years (15.6 per cent, 95 per cent c.i 12.7-18.6 per cent) and 30-39 years (14.2 per cent, 12.2-16.2 per cent) and lowest among those aged 70-79 years (6.8 per cent, 5.7-8.0 per cent). Similar incidence rate increases were reported across all socioeconomic deprivation quintiles and in both sexes. Analysis by grade of NET showed that grade 1 tumours accounted for 63 per cent between 2010 and 2013, compared with 2 per cent between 2000 and 2003. CONCLUSIONS: The incidence rate of malignant appendiceal tumours has increased significantly since 1995 and is mainly attributed to an increase in NETs. The increased diagnosis of low-grade NETs may in part be due to changes in pathological classification systems.