In vitro functional analysis and in silico structural modelling of pathogen-secreted polyglycine hydrolases.

Polyglycine hydrolases are fungal effectors composed of an N-domain with unique sequence and structure and a C-domain that resembles ß-lactamases, with serine protease activity. These secreted fungal proteins cleave Gly-Gly bonds within a polyglycine sequence in corn ChitA chitinase. The polyglycine hydrolase N-domain (PND) function is unknown. In this manuscript we provide evidence that the PND does not directly participate in ChitA cleavage. In vitro analysis of site-directed mutants in conserved residues of the PND of polyglycine hydrolase Es-cmp did not specifically impair protease activity. Furthermore, in silico structural models of three ChitA-bound polyglycine hydrolases created by High Ambiguity Driven protein-protein DOCKing (HADDOCK) did not predict significant interactions between the PND and ChitA. Together these results suggest that the PND has another function. To determine what types of PND-containing proteins exist in nature we performed a computational analysis of Foldseek-identified PND-containing proteins. The analysis showed that proteins with PNDs are present throughout biology as either single domain proteins or fused to accessory domains that are diverse but are usually proteases or kinases.

Developing the E. coli platform for efficient production of UMP-derived chemicals.

5-Methyluridine (5-MU) is a prominent intermediate for industrial synthesis of several antiviral-drugs, however, its availability over the past decades has overwhelmingly relied on chemical and enzymatic strategies. Here, we have realized efficient production of 5-MU in E. coli, for the first time, via a designer artificial pathway consisting of a two-enzyme cascade (UMP 5-methylase and phosphatase). More importantly, we have engineered the E. coli cell factory to boost 5-MU production by systematic evaluation of multiple strategies, and as a proof of concept, we have further developed an antibiotic-free fermentation strategy to realize 5-MU production (10.71 g/L) in E. coli MB229 (a ΔthyA strain). Remarkably, we have also established a versatile and robust platform with exploitation of the engineered E. coli for efficient production of diversified UMP-derived chemicals. This study paves the way for future engineering of E. coli as a synthetic biology platform for acceleratively accessing UMP-derived chemical diversities.

Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis.

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis. While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map.IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis. This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map. This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.

The tunicamycin derivative TunR2 exhibits potent antibiotic properties with low toxicity in an in vivo Mycobacterium marinum-zebrafish TB infection model.

Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes.

Precursor-Directed Biosynthesis and Biological Testing of omega-Alicyclic- and neo-Branched Tunicamycin N-Acyl Variants.

Tunicamycins (TUNs) are Streptomyces-derived natural products, widely used to block protein N-glycosylation in eukaryotes or cell wall biosynthesis in bacteria. Modified or synthetic TUN analogues that uncouple these activities have considerable potential as novel mode-of-action antibacterial agents. Chemically modified TUNs reported previously with attenuated activity on yeast have pinpointed eukaryotic-specific chemophores in the uridyl group and the N-acyl chain length and terminal branching pattern. A small molecule screen of fatty acid biosynthetic primers identified several novel alicyclic- and neo-branched TUN N-acyl variants, with primer incorporation at the terminal omega-acyl position. TUNs with unique 5- and 6-carbon ω-cycloalkane and ω-cycloalkene acyl chains are produced under fermentation and in yields comparable with the native TUN. The purification, structural assignments, and the comparable antimicrobial properties of 15 of these compounds are reported, greatly extending the structural diversity of this class of compounds for potential medicinal and agricultural applications.

Reflecting on insight and insights into reflection: a systematic review of insight and reflection in post graduate medical education.

BACKGROUND: Clinicians recognize insight as important for safe independent medical practice. Clinical education literature focuses on self-reflection. The aim of this review is to describe how clinical educators conceptualize reflection and ask is it analogous to how clinicians conceptualize insight? METHODS: Using PRISMA guidelines, a systematic review of the literature around insight and reflection in postgraduate medical education was undertaken. A thematic analysis of the concepts of insight and reflection was performed. RESULTS: A total of 75 reports were included in the analysis. The literature focussed predominantly on reflection with little discussion of insight. Three main themes were generated: episodic reflection; cyclic reflection; reflection as a state. Reflection as a state seemed to be the professional quality most often aspired to but was less well defined in terms of educational interventions. When more than one model was described, it was often with a reflective state being the ideal that episodic or cyclic reflection may approximate. It is not clear that it is possible to progress up the hierarchy. CONCLUSION: We present a novel description of a hierarchy from discrete episodes of reflection, to cyclic processes that involve reflection, through to a state in which the practitioner is reflective. There is no unified understanding of how an individual ascends this hierarchy, or a cohesive description of what insight is for an independent medical practitioner. This review highlights the need for research into how practicing clinicians conceptualize and characterize insight in their training and practice.

Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

Crystal structure of a polyglycine hydrolase determined using a RoseTTAFold model.

Polyglycine hydrolases (PGHs) are secreted fungal proteases that cleave the polyglycine linker of Zea mays ChitA, a defensive chitinase, thus overcoming one mechanism of plant resistance to infection. Despite their importance in agriculture, there has been no previous structural characterization of this family of proteases. The objective of this research was to investigate the proteolytic mechanism and other characteristics by structural and biochemical means. Here, the first atomic structure of a polyglycine hydrolase was identified. It was solved by X-ray crystallography using a RoseTTAFold model, taking advantage of recent technical advances in structure prediction. PGHs are composed of two domains: the N- and C-domains. The N-domain is a novel tertiary fold with an as-yet unknown function that is found across all kingdoms of life. The C-domain shares structural similarities with class C ß-lactamases, including a common catalytic nucleophilic serine. In addition to insights into the PGH family and its relationship to ß-lactamases, the results demonstrate the power of complementing experimental structure determination with new computational techniques.

Light Stimulates Copper-Limited Growth of an Oceanic Diatom by Increasing Cellular Copper(II) Reduction─A Rate-Determining Step in Copper Uptake.

Uptake of Cu by Thalassiosira oceanica requires that Cu(II) is reduced to Cu(I) prior to transport across the cell membrane. The reduction step is mediated biochemically by cellular reductases active with a broad range of Cu chemical species. Here, we report on the cellular Cu(II) reduction and Cu(I) uptake of a diatom under saturating and subsaturating irradiance. An increase in growth irradiance, from 50 to 400 µmol photons m-2 s-1, increased the rate of extracellular Cu(II) reduction and steady-state Cu uptake. Under these conditions, Cu-limited cells acquired Cu more efficiently and maintained faster rates of growth than Cu-limited cells in low light. Pseudo-first-order reaction rate constants were about 70-fold faster for Cu(I) uptake than for Cu(II) reduction so that reduction was the rate-determining step in Cu acquisition. Accordingly, steady-state Cu uptake rates predicted from the reduction rate constants agreed well with measured rates of Cu uptake obtained from cultures growing at low nanomolar Cu concentrations. Transcript abundance of putative Cu(II) reductases followed a similar pattern to cupric reductase activity, increasing in Cu-limited cells and with increasing growth irradiance. The results are significant in showing Cu(II) reduction as the rate-determining step in Cu uptake: they suggest that biologically mediated Cu(II) reduction may be an important part of the Cu cycle in surface waters of the open sea.

Transcriptomes of an oceanic diatom reveal the initial and final stages of acclimation to copper deficiency.

Copper (Cu) concentration is greatly reduced in the open sea so that phytoplankton must adjust their uptake systems and acclimate to sustain growth. Acclimation to low Cu involves changes to the photosynthetic apparatus and specific biochemical reactions that use Cu, but little is known how Cu affects cellular metabolic networks. Here we report results of whole transcriptome analysis of a plastocyanin-containing diatom, Thalassiosira oceanica 1005, during its initial stages of acclimation and after long-term adaptation in Cu-deficient seawater. Gene expression profiles, used to identify Cu-regulated metabolic pathways, show downregulation of anabolic and energy-yielding reactions in Cu-limited cells. These include the light reactions of photosynthesis, carbon fixation, nitrogen assimilation and glycolysis. Reduction of these pathways is consistent with reduced growth requirements for C and N caused by slower rates of photosynthetic electron transport. Upregulation of oxidative stress defence systems persists in adapted cells, suggesting cellular damage by increased reactive oxygen species (ROS) occurs even after acclimation. Copper deficiency also alters fatty acid metabolism, possibly in response to an increase in lipid peroxidation and membrane damage driven by ROS. During the initial stages of Cu-limitation the majority of differentially regulated genes are associated with photosynthetic metabolism, highlighting the chloroplast as the primary target of low Cu availability. The results provide insights into the mechanisms of acclimation and adaptation of T. oceanica to Cu deficiency.

Efficient biosynthesis of nucleoside cytokinin angustmycin A containing an unusual sugar system.

Angustmycin A has anti-mycobacterial and cytokinin activities, and contains an intriguing structure in which an unusual sugar with C5'-C6' dehydration is linked to adenine via an N-glycosidic bond. However, the logic underlying the biosynthesis of this molecule has long remained obscure. Here, we address angustmycin A biosynthesis by the full deciphering of its pathway. We demonstrate that AgmD, C, A, E, and B function as D-allulose 6-phosphate 3-epimerase, D-allulose 6-phosphate pyrophosphokinase, adenine phosphoallulosyltransferase, phosphoribohydrolase, and phosphatase, respectively, and that these collaboratively catalyze the relay reactions to biosynthesize angustmycin C. Additionally, we provide evidence that AgmF is a noncanonical dehydratase for the final step to angustmycin A via a self-sufficient strategy for cofactor recycling. Finally, we have reconstituted the entire six-enzyme pathway in vitro and in E. coli leading to angustmycin A production. These results expand the enzymatic repertoire regarding natural product biosynthesis, and also open the way for rational and rapid discovery of other angustmycin related antibiotics.

The composition of the founding population of Iceland: A new perspective from 3D analyses of basicranial shape.

The settlement of Iceland in the Viking Age has been the focus of much research, but the composition of the founding population remains the subject of debate. Some lines of evidence suggest that almost all the founding population were Scandinavian, while others indicate a mix of Scandinavians and people of Scottish and Irish ancestry. To explore this issue further, we used three-dimensional techniques to compare the basicrania of skeletons from archaeological sites in Iceland, Scandinavia, and the British Isles. Our analyses yielded two main results. One was that the founding population likely consisted of roughly equal numbers of Scandinavians and people from the British Isles. The other was that the immigrants who originated from the British Isles included individuals of southern British ancestry as well as individuals of Scottish and Irish ancestry. The first of these findings is consistent with the results of recent analyses of modern and ancient DNA, while the second is novel. Our study, therefore, strengthens the idea that the founding population was a mix of Scandinavians and people from the British Isles, but also raises a new possibility regarding the regions from which the settlers originated.

Bilateral Wilms' tumour: An international comparison of treatments and outcomes.

INTRODUCTION: Wilms' tumour is the most common childhood renal malignancy, with 5-10% of cases presenting bilaterally 1. However, there is currently no consensus between centres on optimal management of bilateral Wilms' tumours. This is an international multi-centre case series comparing management and outcomes of bilateral Wilms' tumours between low-income centres (LIC) and high-income centres (HIC). METHODS: Patients with bilateral Wilms' tumour were identified from four tertiary referral centres internationally. Data were collected on baseline characteristics, disease status, treatment used and clinical outcomes. Results were compared between individual centres as well as between groups of low-income centres (LIC) and high-income centres (HIC). RESULTS: Data were collected for forty patients. Most patients received preoperative chemotherapy (n = 38, 95%). The most common surgical procedures were bilateral nephron-sparing surgery (n = 10, 25%) and nephrectomy with partial nephrectomy (n = 20, 50%). Ten-year survival after treatment was as follows: LIC's n = 13 (65%); HIC's n = 20 (100%) (p = 0.01). DISCUSSION: Ten-year survival was significantly higher in HIC's. Our results show this may be caused by patient factors such as later presentation with more advanced disease in low-income centres. This comparative case series is the first to report on a large number of cases from multiple international centres, and to compare key outcomes.

Branched Chain Lipid Metabolism As a Determinant of the N-Acyl Variation of Streptomyces Natural Products.

Branched-chain fatty acids (BCFA) are encountered in Gram-positive bacteria, but less so in other organisms. The bacterial BCFA in membranes are typically saturated, with both odd- and even-numbered carbon chain lengths, and with methyl branches at either the ω-1 (iso) or ω-2 (anteiso) positions. The acylation with BCFA also contributes to the structural diversity of microbial natural products and potentially modulates biological activity. For the tunicamycin (TUN) family of natural products, the toxicity toward eukaryotes is highly dependent upon N-acylation with trans-2,3-unsaturated BCFA. The loss of the 2,3-unsaturation gives modified TUN with reduced eukaryotic toxicity but crucially with retention of the synergistic enhancement of the ß-lactam group of antibiotics. Here, we infer from genomics, mass spectrometry, and deuterium labeling that the trans-2,3-unsaturated TUN variants and the saturated cellular lipids found in TUN-producing Streptomyces are derived from the same pool of BCFA metabolites. Moreover, non-natural primers of BCFA metabolism are selectively incorporated into the cellular lipids of TUN-producing Streptomyces and concomitantly produce structurally novel neo-branched TUN N-acyl variants.

Harnessing synthetic biology-based strategies for engineered biosynthesis of nucleoside natural products in actinobacteria.

Antibiotic resistance poses an increasing threat to global health, and it is urgent to reverse the present trend by accelerating development of new natural product derived drugs. Nucleoside antibiotics, a valuable family of promising natural products with remarkable structural features and diverse biological activities, have played significant roles in healthcare and for plant protection. Understanding the biosynthesis of these intricate molecules has provided a foundation for bioengineering the microbial cell factory towards yield enhancement and structural diversification. In this review, we summarize the recent progresses in employing synthetic biology-based strategies to improve the production of target nucleoside antibiotics. Moreover, we delineate the advances on rationally accessing the chemical diversities of natural nucleoside antibiotics.

Exploring the Active Site of the Antibacterial Target MraY by Modified Tunicamycins.

The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.

Identification of copper-regulated proteins in an oceanic diatom, Thalassiosira oceanica 1005.

Copper (Cu) is an essential cofactor of photosynthetic and respiratory redox proteins in phytoplankton and a scarce resource in parts of the open sea. Although its importance for growth is well recognized, the molecular mechanisms by which phytoplankton respond and acclimate to Cu deficiency are not well known. In this study, we identified the dominant Cu-regulated proteins and measured key physiological traits of Thalassiosira oceanica (CCMP 1005) under Cu-limiting and sufficient conditions. Growth limitation of T. oceanica occurred at environmentally relevant Cu concentrations (1 nM) as a result of decreased photosynthetic efficiency (ΦPSII). In Cu-limited cells, levels of plastocyanin decreased by 3-fold compared to Cu-replete cells and rates of maximum photosynthetic electron transport were reduced. Proteins associated with light harvesting complexes also declined in response to Cu limitation, presumably to adjust to reduced photosynthetic electron flow and to avoid photodamage to the photosystems. Key enzymes involved in carbon and nitrogen assimilation were down-regulated in low-Cu cells, as were steady state rates of C and N uptake. Relatively fewer proteins were up-regulated by Cu limitation, but among them were two enzymes involved in fatty acid oxidation (FAO). The increase in FAO may be a sign of increased turnover of cellular lipids caused by damage from oxidative stress. A putative transcription factor containing three, repetitive methionine motifs (MpgMgggM; MpgMggM) increased significantly in Cu-limited cells. The collective results provide a general description of how plastocyanin-dependent diatoms adjust metabolism to cope with chronic Cu deficiency.

Kilbournase, a protease-associated domain subtilase secreted by the fungal corn pathogen Stenocarpella maydis.

Subtilases are a large family of serine proteases that occur throughout biology. A small subset contain protease-associated (PA) domains that are structurally separate from but encoded within the active site. In bacteria, subtilase PA domains function to recruit specific protein substrates. Here we demonstrate that a protease secreted by the fungal corn pathogen Stenocarpella maydis, which truncates corn ChitA chitinase, is a PA domain subtilase. Protease was purified from S. maydis cultures and tryptic peptides were analyzed by LC-MS/MS. Ions were mapped to two predicted PA domain subtilases. Yeast strains were engineered to express each protease. One failed to produce recombinant protein while the other secreted protease that truncated ChitA. This protease, that we named kilbournase, was purified and characterized. It cleaved multiple peptide bonds in the amino-terminal chitin binding domain of ChitA while leaving the catalytic domain intact. Kilbournase was more active on the ChitA-B73 alloform compared to ChitA-LH82 and did not cleave AtChitIV3, a homolog from Arabidopsis thaliana, indicating a high level of specificity. Truncation of corn ChitA by kilbournase resembles truncation of human C5a by Streptococcus pyogenes ScpA, arguing that PA domain proteases in bacteria and fungi may commonly target specific host proteins.

Quantitative NIR determination of isoflavone and saponin content of ground soybeans.

Over 3200 discrete soybean samples were obtained from production locations around the United States during the years 2012-2016. Ground samples were scanned on near infrared spectrometers (NIRS) and analyzed by HPLC for total isoflavone and total saponin composition, as well as total carbohydrate composition. Multiple Linear Regression (MLR) analysis of preprocessed spectral data was used to develop optimized models to predict isoflavone content. The selection of a suitable calibration model was based on a high regression coefficient (R2), and lower standard error of calibration (SEC) values. Robust validated predictions were obtained for isoflavones, however less than robust calibrations were obtained for the total saponins. The correlations were not as robust for predicting the carbohydrate composition. NIRS is a suitable, rapid, nondestructive method to determine isoflavone composition in ground soybeans. Useful isoflavone composition predictions for large numbers of soybean samples can be obtained from quickly obtained NIRS scans.