Efficacy and Safety of Ketamine Compared with Placebo and Other Medications for Preventing Propofol Injection Pain in Adults: A Systematic Review and Meta-Analysis.

Purpose: To systematically evaluate the effectiveness and safety of ketamine in preventing propofol injection pain (PIP). Patients and Methods: The electronic databases including PubMed, Embase, Web of Science, and Cochrane Library were searched from their inception until 2 August 2023. Randomized controlled trials (RCT) comparing ketamine with placebo or other interventions to alleviate PIP in adults were included. Fixed-effects or random-effects models were used to calculate pooled risk ratios (RR) and corresponding 95% confidence intervals (CI) based on the heterogeneity of the studies included. Results: Thirteen RCTs involving 2105 patients were included. In terms of reducing the incidence of PIP, ketamine is more effective than placebo (RR = 0.43, 95% CI = [0.34, 0.55], P < 0.00001), lidocaine (RR = 0.70, 95% CI = [0.55, 0.90], P = 0.005), dexmedetomidine (RR = 0.52, 95% CI = [0.40, 0.66], P < 0.00001), and thiopental (RR = 0.25, 95% CI = [0.08, 0.83], P = 0.02). In reducing the incidence of severe PIP, ketamine is superior to placebo (RR = 0.12, 95% CI = [0.08, 0.19], P < 0.00001), and lidocaine (RR = 0.34, 95% CI = [0.21, 0.56], P < 0.0001), except dexmedetomidine (RR = 0.20, 95% CI = [0.04, 1.13], P = 0.07), and thiopental (RR = 0.33, 95% CI = [0.04, 3.10], P = 0.33). Compared with mixed injection, separate injection of ketamine and propofol showed no significant difference in the incidence of PIP (RR = 0.96, 95% CI = [0.31, 3.00], P = 0.95) and severe PIP (RR = 1.19, 95% CI = [0.07, 21.29], P = 0.90). Based solely on the reports from the studies included, subanesthetic doses of ketamine are generally safe in preventing PIP. Conclusion: A subanesthetic dose of ketamine can effectively and safely reduce the incidence of PIP and severe PIP in adults, and is more effective than lidocaine, dexmedetomidine, and thiopental. Registration: PROSPERO CRD42023455093.

Comparison of Remimazolam-Flumazenil versus Propofol for Recovery from General Anesthesia: A Systematic Review and Meta-Analysis.

(1) Purpose: to systematically evaluate the recovery following sedation and anesthesia with remimazolam combined with flumazenil in comparison to propofol. (2) Methods: Electronic databases, including PubMed, Embase, Web of Science, and the Cochrane Library, were systematically searched from their inception up to 22 October 2023. Included in this analysis were randomized controlled trials (RCT) that compared remimazolam-flumazenil with propofol for the recovery from sedation and anesthesia in adults. The risk of bias was assessed using the Cochrane risk of bias tool. Pooled risk ratios (RR) or mean differences (MD) along with their corresponding 95% confidence intervals (CI) were calculated using either fixed-effects or random-effects models, and the results were visualized in forest plots. (3) Results: Nine RCTs involving 745 patients who underwent general anesthesia in three different countries were included. Compared to propofol, the remimazolam-flumazenil combination shortened the emergence time (MD = -4.34 min, 95% CI = [-6.88, -1.81], p = 0.0008, low certainty), extubation time (MD = -4.26 min, 95% CI = [-6.81, -1.7], p = 0.0011, low certainty), and the post-anesthesia care unit (PACU) stay (MD = -4.42 min, 95% CI = [-7.45, -1.38], p = 0.0044, low certainty), while reducing the incidence of respiratory depression (RR = 0.2, 95% CI = [0.04, 0.89], p = 0.03, high certainty) after general anesthesia. However, this combination was associated with a higher incidence of re-sedation (RR = 4.15, 95% CI = [1.31, 13.13], p = 0.01, moderate certainty). (4) Conclusions: Based on the existing evidence, the combination of remimazolam and flumazenil accelerates recovery from general anesthesia and lowers the risk of respiratory depression compared to propofol. However, it is important to consider the higher risk of re-sedation when using this combination in clinical practice. Due to limitations in the quality of the evidence, it is advisable to interpret the results of meta-analyses with caution.

Prognostic value of plasma 7-ketocholesterol in sepsis.

BACKGROUND: Early evaluation of the severity of sepsis and estimation of its prognosis remains one of the main challenges in current therapeutic strategies. This study aimed to evaluate the prognostic value of plasma 7-ketocholesterol (7-KC) in sepsis. METHODS: We retrospectively measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) the plasma 7-KC concentration in 176 patients with sepsis and 90 healthy volunteers. A multivariate Cox proportional hazard model was introduced to identify independent factors, including plasma 7-KC and clinical features, for the 28-day mortality of sepsis, and a nomogram for predicting the 28-day mortality of sepsis was established. Decision curve analysis (DCA) was performed to assess the prediction model of death risk of sepsis. RESULTS: The area under the curve (AUC) of plasma 7-KC in diagnosing sepsis was 0.899 (95% CI = 0.862-0.935, P < 0.001), while it was 0.830 (95% CI = 0.764-0.894, P < 0.001) in diagnosing septic shock. The AUCs of plasma 7-KC in predicting the survival of sepsis patients in the training cohort and the test cohort were 0.770 (95% CI = 0.692-0.848, P < 0.05) and 0.869 (95% CI = 0.763-0.974, P < 0.05), respectively. In addition, high plasma 7-KC expression predicts poor prognosis in sepsis. Then, 7-KC and platelet count were identified as the two factors with significant differences by a multivariate Cox proportional hazard model, and the 28-day mortality probability ranged from 0.002 to 0.985 and was assessed using a nomogram. DCA results revealed that the combination of plasma 7-KC and platelet count showed the best prognostic efficiency of the risk threshold compared to a single factor in both the training cohort and test cohort. CONCLUSIONS: Collectively, the elevated plasma 7-KC level is an indicator of sepsis and was identified as a prognostic indicator for sepsis patients, providing a landscape for predicting survival in early sepsis with potential clinical utility.

Characterization of a barley (Hordeum vulgare L.) mutant with multiple stem nodes and spikes and dwarf (msnsd) and fine-mapping of its causal gene.

Introduction: Multiple nodes and dwarf mutants in barley are a valuable resource for identifying genes that control shoot branching, vegetative growth and development. Methods: In this study, physiological, microscopic and genetic analysis were conducted to characterize and fine-map the underling gene of a barley mutant with Multiple Stem Nodes and Spikes and Dwarf (msnsd), which was selected from EMS- and 60Co-treated barley cv. Edamai 934. Results and discussion: The msnsd mutant had more stem nodes, lower plant height and a shorter plastochron than Edamai 934. Moreover, the mutant had two or more spikes on each tiller. Microscopic analysis showed that the dwarf phenotype of msnsd resulted from reduced cell lengths and cell numbers in the stem. Further physiological analysis showed that msnsd was GA3-deficient, with its plant height increasing after external GA3 application. Genetic analysis revealed that a single recessive nuclear gene, namely, HvMSNSD, controlled the msnsd phenotype. Using a segregating population derived from Harrington and the msnsd mutant, HvMSNSD was fine-mapped on chromosome 5H in a 200 kb interval using bulked segregant analysis (BSA) coupled with RNA-sequencing (BSR-seq), with a C-T substitution in the exon of HvTCP25 co-segregating with the msnsd phenotype. RNA-seq analysis showed that a gene encoding gibberellin 2-oxidase 8, a negative regulator of GA biosynthesis, was upregulated in the msnsd mutant. Several known genes related to inflorescence development that were also upregulated and enriched in the msnsd mutant. Collectively, we propose that HvMSNSD regulates the plastochron and morphology of reproductive organs, likely by coordinating GA homeostasis and changed expression of floral development related genes in barley. This study offers valuable insights into the molecular regulation of barley plant architecture and inflorescence development.

Positional cloning identified HvTUBULIN8 as the candidate gene for round lateral spikelet (RLS) in barley (Hordeum vulgare L.).

KEY MESSAGE: Map-based cloning, subcellular localization, virus-induced-gene-silencing and transcriptomic analysis reveal HvTUB8 as a candidate gene with pleiotropic effects on barley spike and leaf development via ethylene and chlorophyll metabolism. Barley lateral spikelet morphology and grain shape play key roles in grain physical quality and yield. Several genes and QTLs for these traits have been cloned or fine mapped previously. Here, we report the phenotypic and genotypic analysis of a barley mutant with round lateral spikelet (rls) from cv. Edamai 934. rls had round lateral spikelet, short but round grain, shortened awn, thick glume and dark green leaves. Histocytologic and ultrastructural analysis revealed that the difference of grain shape of rls was caused by change of cell arrangement in glume, and the dark leaf color resulted from enlarged chloroplast. HvTUBULIN8 (HvTUB8) was identified as the candidate gene for rls by combination of RNA-Seq, map-based-cloning, virus-induced-gene-silencing (VIGS) and protein subcellular location. A single G-A substitution at the third exon of HvTUB8 resulted in change of Cysteine 354 to tyrosine. Furthermore, the mutant isoform Hvtub8 could be detected in both nucleus and cytoplasm, whereas the wild-type protein was only in cytoplasm and granular organelles of wheat protoplasts. Being consistent with the rare phenotype, the "A" allele of HvTUB8 was only detected in rls, but not in a worldwide barley germplasm panel with 400 accessions. VIGS confirmed that HvTUB8 was essential to maintain spike integrity. RNA-Seq results suggested that HvTUB8 may control spike morphogenesis via ethylene homeostasis and signaling, and control leaf color through chlorophyll metabolism. Collectively, our results support HvTUB8 as a candidate gene for barley spike and leaf morphology and provide insight of a novel mechanism of it in barley development.

Molecular Mapping of QTLs Conferring Fusarium Head Blight Resistance in Chinese Wheat Cultivar Jingzhou 66.

Fusarium head blight (FHB) is a destructive disease of wheat (Triticum aestivum L.), which not only significantly reduces grain yield, but also affects end-use quality. Breeding wheat cultivars with high FHB resistance is the most effective way to control the disease. The Chinese wheat cultivar Jingzhou 66 (JZ66) shows moderately high FHB resistance; however, the genetic basis of its resistance is unknown. A doubled haploid (DH) population consisting 209 lines was developed from a cross of JZ66 and Aikang 58 (AK58), a FHB susceptible wheat cultivar, to identify quantitative trait loci (QTL) that contribute to the FHB resistance. Five field experiments were established across two consecutive crop seasons (2018 and 2019) to evaluate the DH lines and parents for FHB response. The parents and DH population were genotyped with the wheat 55K single-nucleotide polymorphism (SNP) assay. Six QTLs associated with FHB resistance in JZ66 were mapped on chromosome 2DS, 3AS, 3AL, 3DL, 4DS, and 5DL, respectively. Four of the QTL (QFhb.hbaas-2DS, QFhb.hbaas-3AL, QFhb.hbaas-4DS, and QFhb.hbaas-5DL) were detected in at least two environments, and the QTL on 3AL and 5DL might be new. The QTL with major effects, QFhb.hbaas-2DS and QFhb.hbaas-4DS, explained up to 36.2% and 17.6% of the phenotypic variance, and were co-localized with the plant semi-dwarfing loci Rht8 and Rht-D1. The dwarfing Rht8 allele significantly increased spike compactness (SC) and FHB susceptibility causing a larger effect on FHB response than Rht-D1 observed in this study. PCR-based SNP markers for QFhb.hbaas-2DS, QFhb.hbaas-3AL, QFhb.hbaas-4DS, and QFhb.hbaas-5DL, were developed to facilitate their use in breeding for FHB resistance by marker-assisted selection.

Discovery of a novel analogue of FR901533 and the corresponding biosynthetic gene cluster from Streptosporangium roseum No. 79089.

FR901533 (1, also known as WS79089B), WS79089A (2), and WS79089C (3) are polycyclic aromatic natural products with promising inhibitory activity to endothelin-converting enzymes. In this work, we isolated five tridecaketide products from Streptosporangium roseum No. 79089, including 1-3, benaphthamycin (4) and a novel FR901533 analogue (5). The structure of 5 was characterized based on spectroscopic data. Compared with the major product 2, the new compound 5 has an additional hydroxyl group at C-12 and an extra methyl group at the 13-OH. The configuration of C-19 of these compounds was determined to be R using Mosher's method. A putative biosynthetic gene cluster for compounds 1-5 was discovered by analyzing the genome of S. roseum No. 79089. This 38.6-kb gene cluster contains 38 open reading frames, including a minimal polyketide synthase (wsaA-C), an aromatase (wsaD), three cyclases (wsaE, F, and W), and a series of tailoring enzymes such as monooxygenases (wsaO1-O7) and methyltransferases (wsaM1 and M2). Disruption of the ketosynthase gene (wsaA) in this gene cluster abolished the production of 1-5, confirming that this gene cluster is indeed responsible for the biosynthesis of 1-5. A type II polyketide biosynthetic pathway was proposed for this group of natural endothelin-converting enzyme inhibitors. KEY POINTS: • Five aromatic tridecaketides were isolated from Streptosporangium roseum No. 79089. • A novel FR901533 analogue, 12-hydroxy-13-O-methyl-WS79089A, was characterized. • The absolute configuration of C-19 of FR901533 and analogues was determined. • The biosynthetic gene cluster of FR901533 and analogues was discovered.

Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase.

BACKGROUND: Creating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture. RESULTS: The MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and N-desmethylbassianolide was produced in low yields. Co-expression of BSLS-ΔMT with standalone MT did not recover bassianolide biosynthesis. In order to address the functional implications of the protein insertion, we characterized the N-methyltransferase activity of the MT domain as both the isolated domain (MTBSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MTBSLS construct demonstrated a relaxed substrate specificity and preferentially methylated an amino acid (L-Phe-SNAC) that is rarely incorporated into the final product. By testing the preference of a series of MT constructs (BSLS, MTBSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MTBSLS by crosslinking or embedding the MT within an A domain narrowed the substrate specificity of the methyltransferase toward L-Leu-SNAC, the preferred substrate for the BSLS megaenzyme. CONCLUSIONS: The embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products.

Human exposure pathways to organophosphate flame retardants: Associations between human biomonitoring and external exposure.

Organophosphate flame retardants (PFRs) have largely replaced the market of polybrominated diphenyl ethers (PBDEs). Concerns about PFR contamination and its impact on human health have consequently increased. A comprehensive investigation on the human exposure pathways to PFRs is to be endeavoured. This study investigated the occurrence of PFR metabolites in human urine, serum and hair, correlating them with external exposure data that was presented in our previous studies. Participants from Oslo (n = 61) provided a set of samples, including dust, air, handwipes, food, urine, serum and hair. Associations between PFR metabolites analyzed in the biological samples and the PFRs in environmental samples were explored. Different sampling strategies for dosimeters (e.g. floor/surface dust, personal/stationary air) were also compared to understand which is better for predicting human exposure to PFRs. Seven out of the eleven target PFR metabolites, including diphenyl phosphate (DPHP) and bis(1-chloro-2-propyl)-1-hydroxy-2-propyl phosphate (BCIPHIPP), were frequently detected (DF > 30%) in urine. DPHP was the most frequently detected metabolite in both serum and hair. Several PFR metabolites had higher levels in morning urine than in afternoon urine. Floor dust appeared to be a better proxy for estimating PFR internal exposure than surface dust, air, and handwipes. Some PFRs in handwipes and air were also correlated with their metabolites in urine and hair. Age, beverage consumption and food consumption were negatively associated with DPHP levels in urine. Discrepancies observed between the external and internal exposure for some PFRs call for further investigation on PFR bioaccessibility and clearance.

An efficient process for co-production of γ-aminobutyric acid and probiotic Bacillus subtilis cells.

This study was to establish an integrated process for the co-production of γ-aminobutyric acid (GABA) and live probiotics. Six probiotic bacteria were screened and Bacillus subtilis ATCC 6051 showed the highest GABA-producing capacity. The optimal temperature and initial pH value for GABA production in B. subtilis were found to be 30 °C and 8.0, respectively. A variety of carbon and nitrogen sources were tested, and potato starch and peptone were the preferred carbon and nitrogen sources for GABA production, respectively. The concentrations of carbon source, nitrogen source and substrate (sodium l-glutamate) were then optimized using the response surface methodology. The GABA titer and concentration of viable cells of B. subtilis reached 19.74 g/L and 6.0 × 108 cfu/mL at 120 h. The GABA titer represents the highest production of GABA in B. subtilis. This work thus demonstrates a highly efficient co-production process for GABA and probiotic B. subtilis cells.

Identification and heterologous reconstitution of a 5-alk(en)ylresorcinol synthase from endophytic fungus Shiraia sp. Slf14.

A new type III polyketide synthase gene (Ssars) was discovered from the genome of Shiraia sp. Slf14, an endophytic fungal strain from Huperzia serrata. The intron-free gene was cloned from the cDNA and ligated to two expression vectors pET28a and YEpADH2p-URA3 for expression in Escherichia coli BL21(DE3) and Saccharomyces cerevisiae BJ5464, respectively. SsARS was efficiently expressed in E. coli BL21(DE3), leading to the synthesis of a series of polyketide products. Six major products were isolated from the engineered E. coli and characterized as 1,3-dihydroxyphenyl-5-undecane, 1,3-dihydroxyphenyl-5-cis-6'-tridecene,1,3-dihydroxyphenyl-5-tridecane, 1,3-dihydroxyphenyl-5-cis-8'-pentadecene, 1,3-dihydroxyphenyl-5-pentadecane, and 1,3-dihydroxyphenyl-5-cis-10'-heptadecene, respectively, based on the spectral data and biosynthetic origin. Expression of SsARS in the yeast also led to the synthesis of the same polyketide products, indicating that this enzyme can be reconstituted in both heterologous hosts. Supplementation of soybean oil into the culture of E. coli BL21(DE3)/SsARS increased the production titers of 1-6 and led to the synthesis of an additional product, which was identified as 5-(8'Z,11'Z-heptadecadienyl) resorcinol. This work thus allowed the identification of SsARS as a 5-alk(en)ylresorcinol synthase with flexible substrate specificity toward endogenous and exogenous fatty acids. Desired resorcinol derivatives may be synthesized by supplying corresponding fatty acids into the culture medium.

Simultaneous determination of 14 urinary biomarkers of exposure to organophosphate flame retardants and plasticizers by LC-MS/MS.

Organophosphate flame retardants and plasticizers (PFRs) are a group of chemicals widely added to consumer products. PFRs are quickly metabolized in the human body into two types of metabolites, (1) dialkyl and diaryl phosphate esters (DAPs), such as diphenyl phosphate (DPHP) and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP); and (2) hydroxylated PFRs (HO-PFRs), such as 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP) and 2-hydroxyethyl bis(2-butoxyethyl) phosphate (BBOEHEP). Existing analytical methods usually focus on DAPs; therefore, human biomonitoring data on HO-PFRs remain scarce. In this study, an analytical procedure was developed for the simultaneous quantification of multiple PFR metabolites in human urine, covering eight DAPs and six HO-PFRs. Sample preparation was optimized to include all target compounds using Bond-Elut C18 solid-phase extraction cartridges, followed by instrumental analysis based on liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS). Method performance was validated according to established guidelines and satisfactory results were obtained for all metabolites in terms of recovery, linearity, limits of quantification, precision, and accuracy. Recoveries ranged from 87 to 112%. Method detection limits from 0.002 ng/mL for 2-ethyl-5-hydroxyhexyl diphenyl phosphate (5-HO-EHDPHP) to 0.66 ng/mL for 4-hydroxyphenyl phenyl phosphate (4-HO-DPHP). Seven PFR metabolites were frequently detected in a small biomonitoring study (n = 14), among them bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), di-n-butyl phosphate (DNBP), 5-HO-EHDPHP, and BBOEHEP. Highest mean concentrations were found for DPHP, 2-ethylhexyl phenyl phosphate (EHPHP), and BCIPHIPP, while 4-HO-DPHP, 5-HO-EHDPHP, and EHPHP were detected in urine for the first time. Overall, the obtained results demonstrate that the developed method can be used for the simultaneous determination of 14 urinary biomarkers of exposure to PFRs. Graphical abstract ᅟ.

Multi-pathway human exposure assessment of phthalate esters and DINCH.

Phthalate esters are substances mainly used as plasticizers in various applications. Some have been restricted and phased out due to their adverse health effects and ubiquitous presence, leading to the introduction of alternative plasticizers, such as DINCH. Using a comprehensive dataset from a Norwegian study population, human exposure to DMP, DEP, DnBP, DiBP, BBzP, DEHP, DINP, DIDP, DPHP and DINCH was assessed by measuring their presence in external exposure media, allowing an estimation of the total intake, as well as the relative importance of different uptake pathways. Intake via different uptake routes, in particular inhalation, dermal absorption, and oral uptake was estimated and total intake based on all uptake pathways was compared to the calculated intake from biomonitoring data. Hand wipe results were used to determine dermal uptake and compared to other exposure sources such as air, dust and personal care products. Results showed that the calculated total intakes were similar, but slightly higher than those based on biomonitoring methods by 1.1 to 3 times (median), indicating a good understanding of important uptake pathways. The relative importance of different uptake pathways was comparable to other studies, where inhalation was important for lower molecular weight phthalates, and negligible for the higher molecular weight phthalates and DINCH. Dietary intake was the predominant exposure route for all analyzed substances. Dermal uptake based on hand wipes was much lower (median up to 2000 times) than the total dermal uptake via air, dust and personal care products. Still, dermal uptake is not a well-studied exposure pathway and several research gaps (e.g. absorption fractions) remain. Based on calculated intakes, the exposure for the Norwegian participants to the phthalates and DINCH was lower than health based limit values. Nevertheless, exposure to alternative plasticizers, such as DPHP and DINCH, is expected to increase in the future and continuous monitoring is required.

Probing the relationship between external and internal human exposure of organophosphate flame retardants using pharmacokinetic modelling.

Human external exposure (i.e. intake) of organophosphate flame retardants (PFRs) has recently been quantified, but no link has yet been established between external and internal exposure. In this study, we used a pharmacokinetic (PK) model to probe the relationship between external and internal exposure data for three PFRs (EHDPHP, TNBP and TPHP) available for a Norwegian cohort of 61 individuals from 61 different households. Using current literature on metabolism of PFRs, we predicted the metabolite serum/urine concentrations and compared it to measured data from the study population. Unavailable parameters were estimated using a model fitting approach (least squares method) after assigning reasonable constraints on the ranges of fitted parameters. Results showed an acceptable comparison between PK model estimates and measurements (<10-fold deviation) for EHDPHP. However, a deviation of 10-1000 was observed between PK model estimates and measurements for TNBP and TPHP. Sensitivity and uncertainty analysis on the PK model revealed that EHDPHP results showed higher uncertainty than TNBP or TPHP. However, there are indications that (1) current biomarkers of exposure (i.e. assumed metabolites) for TNBP and TPHP chemicals might not be specific and ultimately affecting the outcome of the modelling and (2) some exposure pathways might be missing. Further research, such as in vivo laboratory metabolism experiments of PFRs including identification of better biomarkers will reduce uncertainties in human exposure assessment.

New insights into pradimicin biosynthesis revealed by two O-methyltransferases.

Pradimicins are a group of antiviral and antifungal natural products from Actinomadura hibisca. Two putative O-methyltransferase genes, pdmF and pdmT, are present in the pradimicin biosynthetic gene cluster. However, there is only one methoxy group (11-OCH3) in pradimicins. Through heterologous expression and in vitro reactions with various substrates, PdmF was characterized as the C-11 O-methyltransferase with a relatively broad substrate specificity. To probe the role of PdmT in pradimicin biosynthesis, the corresponding gene was disrupted through homologous recombination, leading to the production of pradimicinone II. This enzyme was then expressed in Escherichia coli with an N-terminal His6 tag and purified by Ni-NTA chromatography. Reaction of pradimicinone II with PdmT generated 7-O-methylpradimicinone II, confirming that this enzyme is a C-7 O-methyltransferase. Characterization of PdmT suggests a novel pathway that leads to the "flip" of 7-OH to C-14 in pradimicin biosynthesis.

Decoding and reprogramming fungal iterative nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) assemble a large group of structurally and functionally diverse natural products. While the iterative catalytic mechanism of bacterial NRPSs is known, it remains unclear how fungal NRPSs create products of desired length. Here we show that fungal iterative NRPSs adopt an alternate incorporation strategy. Beauvericin and bassianolide synthetases have the same C1-A1-T1-C2-A2-MT-T2a-T2b-C3 domain organization. During catalysis, C3 and C2 take turns to incorporate the two biosynthetic precursors into the growing depsipeptide chain that swings between T1 and T2a/T2b with C3 cyclizing the chain when it reaches the full length. We reconstruct the total biosynthesis of beauvericin in vitro by reacting C2 and C3 with two SNAC-linked precursors and present a domain swapping approach to reprogramming these enzymes for peptides with altered lengths. These findings highlight the difference between bacterial and fungal NRPS mechanisms and provide a framework for the enzymatic synthesis of non-natural nonribosomal peptides.

Assessment of dietary exposure to organohalogen contaminants, legacy and emerging flame retardants in a Norwegian cohort.

Polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs), emerging halogenated flame retardants (EHFRs) and organophosphate flame retardants (PFRs) were detected in 24h duplicate diet samples from a Norwegian cohort (n=61), with concentrations ranging from

Legacy and alternative flame retardants in Norwegian and UK indoor environment: Implications of human exposure via dust ingestion.

Indoor dust has been acknowledged as a major source of flame retardants (FRs) and dust ingestion is considered a major route of exposure for humans. In the present study, we investigated the presence of PBDEs and alternative FRs such as emerging halogenated FRs (EHFRs) and organophosphate flame retardants (PFRs) in indoor dust samples from British and Norwegian houses as well as British stores and offices. BDE209 was the most abundant PBDE congener with median concentrations of 4700ngg-1 and 3400ngg-1 in UK occupational and house dust, respectively, 30 and 20 fold higher than in Norwegian house dust. Monomeric PFRs (m-PFRs), including triphenyl phosphate (TPHP), tris(chloropropyl) phosphate (TCPP) and tris(2-chloroethyl) phosphate (TCEP) dominated all the studied environments. To the best of our knowledge, this is the first report of isodecyldiphenyl phosphate (iDPP) and trixylenyl phosphate (TXP) in indoor environments. iDPP was the most abundant oligomeric PFR (o-PFR) in all dust samples, with median concentrations one order of magnitude higher than TXP and bisphenol A bis(diphenyl phosphate (BDP). iDPP and TXP worst-case scenario exposures for British workers during an 8h exposure in the occupational environment were equal to 34 and 1.4ngkgbw-1day-1, respectively. The worst-case scenario for BDE209 estimated exposure for British toddlers (820ngkgbw-1day-1) did not exceeded the proposed reference dose (RfD) (7000ngkgbw-1day-1), while exposures for sum of m-PFRs (Σm-PFRs) in British toddlers and adults (17,900 and 785ngkgbw-1day-1 respectively) were an order of magnitude higher than for Norwegian toddlers and adults (1600 and 70ngkgbw-1day-1).

Identification of Cyclic Depsipeptides and Their Dedicated Synthetase from Hapsidospora irregularis.

Seven cyclic depsipeptides were isolated from Hapsidospora irregularis and structurally characterized as the calcium channel blocker leualacin and six new analogues based on the NMR and HRESIMS data. These new compounds were named leualacins B-G. The absolute configurations of the amino acids and 2-hydroxyisocaproic acids were determined by recording the optical rotation values. Biological studies showed that calcium influx elicited by leualacin F in primary human lobar bronchial epithelial cells involves the TRPA1 channel. Through genome sequencing and targeted gene disruption, a noniterative nonribosomal peptide synthetase was found to be involved in the biosynthesis of leualacin. A comparison of the structures of leualacin and its analogues indicated that the A2 and A4 domains of the leualacin synthetase are substrate specific, while A1, A3, and A5 can accept alternative precursors to yield new molecules.