Search

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. Abstract 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.

Barley lateral spikelet morphology and grain shape play a key role 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 c.v. Edamai 934. The mutant 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 functional 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 in exon of HvTUB8 resulted in change of Cysteine to Tyrosine. Furthermore, the mutant isoform of HvTUB8 could be detected in both nucleus and cytoplasm, whereas the wild was only in cytoplasm 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 consisting about 400 accessions. VIGS confirmed that HvTUB8 was essential to maintain spike integrity. RNA-sequencing results suggest that HvTUB8 control spike morphogenesis via ethylene homeostasis and signaling, and control leaf color through chlorophyll metabolism. Collectively, our results demonstrated that HvTUB8 has pleiotropic effects on barley spike and leaf morphology and provided insight of a novel mechanism of HvTUB8 in spike and leaf development.

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.

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.

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.

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.

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 HR-ESI-MS 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 non-iterative 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.

Figure S1. ESI-MS (+) spectra of the demethylated nonribosomal peptides. (A) N-Desmethylbassianolide. (B) N-Desmethylbeauvericin. (C) N-Desmethylbeauvericin A. (D) N-Desmethylbeauvericin B. Figure S2. 1H NMR spectrum of N-desmethylbeauvericin. Figure S3. 13C NMR spectrum of N-desmethylbeauvericin. Figure S4. Selected 1H-1H COSY and HMBC corrections for N-desmethylbeauvericin. Figure S5. HPLC analysis (210 nm) of products from the co-expression of isolated MT domain with MT-removed BSLS/MT-inactivated BEAS in S. cerevisiae. (A) Co-expression of MT(BSLS) with BSLS-ΔMT. (B) Co-expression of MT(BEAS) with BEAS-G2131A. Figure S6. ESI-MS(+) spectrum of the N-methylated products of L-Ile-SNAC (A) and L-Val-SNAC. Figure S7. Location of residues in MTBSLS that are proposed to interact with the amino acid substrate. The active site residues (red) of the MT domain in TioS(A4aM4A4b) (pdb ID 5wmm) (gray) that are proposed to interact with the valine side chain (magenta) of the enzyme-bound substrate (G525, W526, M540, W543, S632, Q635, D664, R666, and L738) were mapped onto the homology model of MTBSLS (blue, with limon amino acid side chains). AdoHcy is shown in green and the tethered substrate for TioS(A4aM4A4b) is shown in dark gray. Figure S8. Architecture of the aMT construct. Adenylation domains consist of one polypeptide containing a large subunit (light gray) and a small subunit (dark gray). The active site exists between the two subunits. The “aMT” construct used in these studies contains the entire MT domain and the small subunit of the adenylation domain. Figure 6a was used in the preparation of this figure. (DOCX 1951 kb)

Rdc2 is a flavin-dependent halogenase from Pochonia chlamydosporia . Through the introduction of a His 6 -tag to both the N- and C-termini, the isolation yield of Rdc2 from Escherichia coli using Ni-NTA affinity chromatography was increased by three-fold. In vitro reaction of Rdc2 and a flavin reductase (Fre) with seven different hydroxyquinolines revealed that 3-hydroxyquinoline ( 3 ), 5-hydroxyquinoline ( 5 ), 6-hydroxyquinoline ( 6 ), and 7-hydroxyquinoline ( 7 ) can be specifically halogenated. These products were prepared by incubating the corresponding substrates with IPTG-induced E. coli BL21(DE3)/Rdc2. They were respectively characterized as 3-hydroxy-4-chloroquinoline ( 3a ), 5-hydroxy-6-chloroquinoline ( 5a ), 5-chloro-6-hydroxyquinoline ( 6a ), and 7-hydroxy-8-chloroquinoline ( 7a ) by NMR and MS analyses. This work represents the first enzymatic preparation of chlorohydroxyquinolines and provides a ‘green’ method to synthesize this group of medicinally important compounds.

This study investigated the contamination status of 21 emerging flame retardants (FRs) in soils (n = 32) and river sediments (n = 8) from an e-waste recycling (EWR) site in the northern part of Vietnam. Among analyzed FRs, higher levels of decabromodiphenyl ethane (DBDPE) (ND–4200 ng/g dw), 1,2-bis-(2,4,6-tribromophenoxy)ethane (BTBPE) (ND–350 ng/g dw) and Dechlorane Plus isomers (DPs) (ND–65 ng/g dw) were found in soils near EWR workshops and open burning places. The highest concentrations of DBDPE (20 ng/g dw), BTBPE (5.7 ng/g dw) and DPs (6.7 ng/g dw) were also detected in sediments collected from the middle of the EWR site. The levels decreased concomitantly with increasing distance from the EWR site. These results indicate that these FRs were released to the surrounding environment from improper recycling activities, such as manual dismantling of devices and open burning of e-wastes. Moreover, the estimated daily intakes of those FRs via soil ingestion were approximately ten times higher for children than adults. To our knowledge, this is a first comprehensive study on characterization of soil and sediment contamination by a series of emerging FRs at an EWR site in Vietnam.

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 × 10(8) 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.

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.

Particle size is a significant parameter which determines the environmental fate and the behavior of dust particles and, implicitly, the exposure risk of humans to particle-bound contaminants. Currently, the influence of dust particle size on the occurrence and seasonal variation of hexabromocyclododecanes (HBCDs) remains unclear. While HBCDs are now restricted by the Stockholm Convention, information regarding HBCD contamination in indoor dust in China is still limited. We analyzed composite dust samples from offices (n = 22), hotels (n = 3), kindergartens (n = 2), dormitories (n = 40), and main roads (n = 10). Each composite dust sample (one per type of microenvironment) was fractionated into 9 fractions (F1-F9: 2000-900, 900-500, 500-400, 400-300, 300-200, 200-100, 100-74, 74-50, and

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. Keywords: Phthalate esters, DPHP, DINCH, Plasticizers, External exposure

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 (

Light harvesting chlorophyll a/b binding proteins (LHC) play an important role in photosynthesis and development of plant. However, limited data is available for this important gene family in barley, especially for their biological functions. In this study, sequences of rice and Arabidopsis LHCs were used as queries to identify the respective orthologues from available barley genomic database. Total 17 non-reduntant HvLHCs were identified. Genomic sequences of these genes were ranged from 780 bp to 2779 bp in length, and ORF sequence of 738 bp to 933 bp. The phylogenetic relationship of members from barley, rice and Arabidopsis revealed that most of them were common to the three species, while some of them were specific to Arabidopsis or the poaceae family. Cis-element analysis showed that along with the universal transcription initiation and enhancement relevant elements, there were also light as well as biotic and abiotic stresses responsive elements in promoter regions of these HvLHCs. Additionally, these genes exhibited quite similar expression pattern during the development of barley plant according to the public normalized RNA-seq and barley array datasets, but responded distinctively to different treatments including dark, heat, salinity and drought, which were confirmed by quantitative real-time PCR analysis. This is the first report of identification of HvLHCs at the genome level, and results presented here would be useful not only for determining the precise role of HvLHCs during barley development and abiotic stress responses, but also for using them in molecular breeding of barley varieties with high yield or high tolerance to stresses.

The anti-cholesterol natural product herboxidiene is synthesized by a noniterative modular polyketide synthase (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG) in Streptomyces chromofuscus A7847. In this work, the putative monooxygenase HerG was expressed in Escherichia coli and the purified enzyme was subjected to biochemical studies. It was identified as a cytochrome P450 enzyme responsible for the stereospecific hydroxylation at C-18. This enzyme is highly substrate-specific as it efficiently hydroxylates 18-deoxy-25-demethyl-herboxidiene, but showed no activity towards 18-deoxy-herboxidiene. The kcat/Km value for the HerG-catalyzed hydroxylation of 18-deoxy-25-demethyl-herboxidiene was determined to be 1669.70±47.36 M(-1) s(-1). In vitro co-reaction of HerG with the methyltransferase HerF and analysis of the product formation in S. chromofuscus A7847 revealed that the biosynthetic intermediate 18-deoxy-25-demethyl-herboxidiene is successively hydroxylated at C-18 by HerG and methylated at 17-OH to yield the final product herboxidiene. The minor metabolite 18-deoxy-hereboxidiene is a byproduct of the biosynthetic pathway.

Dust samples were collected in Beijing, China, from four different indoor microenvironments (office, hotel, kindergarten, and student dormitory) and one outdoor (road dust) microenvironment. These five composite samples were fractionated into 13 sequential size fractions and an individual fraction of

This study documents the temporal variability in concentrations of flame retardants (FRs) in floor dust from three offices in Beijing, China. Dust from Office A (OAD) was collected weekly from March to August, 2012, and sampling of dust from Office B and C (OBD and OCD) was conducted fortnightly (each two weeks) from March to December 2012. With intensive and continuous sampling, we report for the first time on clear and coherent temporal trends of polybrominated diphenyl ethers (PBDEs), novel brominated flame retardants (NBFRs) and phosphorus flame retardants (PFRs) in indoor dust. The observed mean concentrations of ∑9PBDEs, ∑4NBFRs and ∑9PFRs, were 554, 11,100 and 128,000 ng g−1 in OAD; 7560, 5000 and 17,300 ng g−1 in OBD; and 4750, 3550 and 17,200 ng g−1 in OCD, respectively. With exception of PBDEs, concentrations of FRs were elevated in OAD than in OBD and OCD. Two to ten-fold variations were observed between the minimum and maximum concentrations of FRs in the same office, indicating that the sampling moment exerts a substantial influence on the level of FR contamination. Different seasonality was distinctively found between BFRs and PFRs. Except for a few occasional abnormal values, BFR levels in office dust were generally constant among different seasons. The abundance rank order for PFRs was: winter > autumn > summer, with peak values occurring in late winter and early spring. This pattern may be attributable to the fact that PFRs are more sensitive to temperature changes compared to PBDEs and NBFRs owning to their higher volatilities. The absence of significant seasonal variation for BFR concentrations in indoor dust compared to outdoor air and dust concentrations is also discussed. Keywords: BFRs, PFRs, Office dust, Seasonal variation, Indoor environment

The herboxidiene biosynthetic gene cluster contains a regulatory gene and six biosynthetic genes that encode three polyketide synthases (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG). Through single crossover recombination, an integrative plasmid was inserted into the genome of Streptomyces chromofuscus ATCC 49982 between herE and herF, resulting in low-level expression of herF and the downstream herG. The mutant strain produced two new compounds, 18-deoxy-25-demethyl-herboxidiene and 25-demethyl-herboxidiene. HerF was expressed in Escherichia coli and biochemically characterized as the dedicated methyltransferase in herboxidiene biosynthesis. It prefers 25-demethyl-herboxidiene to 18-deoxy-25-demethyl-herboxidiene, suggesting that C-25 methylation is the last tailoring step.

A putative indigoidine biosynthetic gene cluster was located in the genome of Streptomyces chromofuscus ATCC 49982. The silent 9.4-kb gene cluster consists of five open reading frames, named orf1, Sc-indC, Sc-indA, Sc-indB, and orf2, respectively. Sc-IndC was functionally characterized as an indigoidine synthase through heterologous expression of the enzyme in both Streptomyces coelicolor CH999 and Escherichia coli BAP1. The yield of indigoidine in E. coli BAP1 reached 2.78 g/l under the optimized conditions. The predicted protein product of Sc-indB is unusual and much larger than any other reported IndB-like protein. The N-terminal portion of this enzyme resembles IdgB and the C-terminal portion is a hypothetical protein. Sc-IndA and/or Sc-IndB were co-expressed with Sc-IndC in E. coli BAP1, which demonstrated the involvement of Sc-IndB, but not Sc-IndA, in the biosynthetic pathway of indigoidine. The yield of indigoidine was dramatically increased by 41.4 % (3.93 g/l) when Sc-IndB was co-expressed with Sc-IndC in E. coli BAP1. Indigoidine is more stable at low temperatures.

Sub-aperture stitching is an effective way to extend the lateral and vertical dynamic range of a conventional interferometer. The test accuracy can be achieved by removing the error of reference surface by the absolute testing method. When the testing accuracy (repeatability and reproducibility) is close to 1nm, in addition to the reference surface, other factors will also affect the measuring accuracy such as environment, zoom magnification, stitching precision, tooling and fixture, the characteristics of optical materials and so on. In the thousand level cleanroom, we establish a good environment system. Long time stability, temperature controlled at 22°±0.02°.The humidity and noise are controlled in a certain range. We establish a stitching system in the clean room. The vibration testing system is used to test the vibration. The air pressure testing system is also used. In the motion system, we control the tilt error no more than 4 second to reduce the error. The angle error can be tested by the autocollimator and double grating reading head.

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., Nonribosomal peptides are important bioactive molecules that are synthetized by enzymes containing several catalytic domains. Here the authors describe the catalytic mechanism of fungal nonribosomal peptide synthetases and present an approach to modify these enzymes to produce specific nonribosomal peptides.

Polyketides represent an important class of biologically active and structurally diverse compounds in nature. They are synthesized from acyl-coenzyme A substrates by polyketide synthases (PKSs). PKSs are classified into three groups: types I, II, and III. This article introduces recent studies on type III PKSs identified from plants, bacteria, and fungi, and describes the catalytic functions of these enzymes in detail. Plant type III PKSs have been widely studied, as exemplified by chalcone synthase, which plays an important role in the synthesis of plant metabolites. Bacterial type III PKSs fall into five groups, many of which were identified from Streptomyces, a genus that has been well known for its production of bioactive molecules and genetic alterability. Although it was believed that type III PKSs exist exclusively in plants and bacteria, recent fungal genome sequencing projects and biochemical studies revealed the presence of type III PKSs in filamentous fungi, which provides a new chance to study fungal secondary metabolism and synthesize “unnatural” natural products. Type III PKSs have been used for the biosynthesis of novel molecules through precursor-directed and structure-based mutagenesis approaches. © 2012 IUBMB Life, 64(4): 285–295, 2012

Lateral shearing interferometry is an attractive technique to measure the wavefront aberration of high numerical aperture optical systems, of which using two-dimensional grating can divide and shear the wavefront in two-dimension simultaneously. A two-dimension lateral shearing interferometer based on chessboard grating was designed, which can work in dual-mode: the phase shifting mode and the Fourier transform mode. In the phase shifting mode, the phase shifting was realized by moving chessboard grating along the shearing direction in the image plane. In the Fourier transform mode, the spatial carrier frequency was realized by positioning the grating at the Talbot distance of the objective image plane. An experimental setup was designed to measure a 10×, NA0.25 microscope objective at 632.8nm wavelength. The objective was measured by the experimental setup in dual-mode, the results showed that the wavefront of the objective was 0.172λ RMS; in the phase shifting mode, the repeatability (3σ) of RMS was 1.1mλ; in the Fourier transform mode, the repeatability (3σ) of RMS was 2.7mλ; after correcting the coordinates of the wavefront, the differences of Z5 to Z36 between phase shifting mode and the Fourier transform mode were better than 8mλ.

Most of the commercial available sub-aperture stitching interferometers measure the surface with a standard lens that produces a reference wavefront, and the precision of the interferometer is generally limited by the standard lens. The test accuracy can be achieved by removing the error of reference surface by the absolute testing method. When the testing accuracy (repeatability and reproducibility) is close to 1nm, in addition to the reference surface, other factors will also affect the measuring accuracy such as environment, zoom magnification, stitching precision, tooling and fixture, the characteristics of optical materials and so on. We establish a stitching system in the thousand level cleanroom. The stitching system is including the Zygo interferometer, the motion system with Bilz active isolation system at level VC-F. We review the traditional absolute flat testing methods and emphasize the method of rotation-shift functions. According to the rotation-shift method we get the profile of the reference lens and the testing lens. The problem of the rotation-shift method is the tilt error. In the motion system, we control the tilt error no more than 4 second to reduce the error. In order to obtain higher testing accuracy, we analyze the influence surface shape measurement accuracy by recording the environment error with the fluke testing equipment.

Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH4)2SO4, NH4Cl, (NH4)2HPO4 and KNO3, (NH4)2HPO4 showed the best effect on improving the titer of indigoidine. Different concentrations of (NH4)2HPO4 were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH4)2HPO4. This work provides two efficient methods for the production of this promising blue pigment in E. coli.

Large-aperture optical elements are widely employed in high-power laser system, astronomy, and outer-space technology. Sub-aperture stitching is an effective way to extend the lateral and vertical dynamic range of a conventional interferometer. Most of the commercial available sub-aperture stitching interferometers measure the surface with a standard lens that produces a reference wavefront, and the precision of the interferometer is generally limited by the standard lens. The test accuracy can be achieved by removing the error of reference surface by the absolute testing method. In our paper we use the different sub-apertures as the different flats to get the profile of the reference lens. Only two lens in the testing process which is fewer than the traditional 3-flat method. In the testing equipment, we add a reflective lens and a lens which can transparent and reflect to get the non rationally symmetric errors of the testing flat. The arithmetic is present in this paper which uses the absolute testing method to improve the testing accuracy of the sub-aperture stitching interferometers by removing the errors caused by reference surface.

A multi-residue analytical method was developed for the determination of a range of flame retardants (FRs), including polybrominated diphenyl ethers (PBDEs), emerging halogenated FRs (EFRs) and organophosphate FRs (PFRs), in food matrices. An ultrasonication and vacuum assisted extraction (UVAE), followed by a multi-stage clean-up procedure, enabled the removal of up to 1. g of lipid from 2.5. g of freeze-dried food samples and significantly reduce matrix effects. UVAE achieves a waste factor (WF) of about 10%, while the WFs of classical QuEChERS methods range usually between 50 and 90%. The low WF of UVAE leads to a dramatic improvement in the sensitivity along with saving up to 90% of spiking (internal) standards. Moreover, a two-stage clean-up on Florisil and aminopropyl silica was introduced after UVAE, for an efficient removal of pigments and residual lipids, which led to cleaner extracts than normally achieved by dispersive solid phase extraction (d-SPE). In this way, the extracts could be concentrated to low volumes, e.g.

Several classes of flame retardants, such as polybrominated diphenyl ethers (PBDEs), novel brominated flame retardants (NBFRs), dechlorane plus (DPs), and organophosphate flame retardants (PFRs), together with polychlorinated biphenyls (PCBs) were measured in indoor dust from five villages located in three e-waste recycling regions in Guangdong Province, South China. The medians of PBDEs, NBFRs, and PFRs in dust in five sites ranged from 685–67,500, 1460–50,010, and 2180–29,000 ng/g, respectively. These concentrations were much higher than the medians of PCBs (52–2900 ng/g). BDE 209 and decabromodiphenyl ethane (DBDPE) were the two major halogen flame retardants in dust, while tris-(1-chloro-2-propyl) phosphate (TCIPP) and triphenyl phosphate (TPHP) were the major PFRs. Principle component analysis revealed the different pollutant patterns among different sites. The estimated median human exposures of PBDEs, NBFRs, PFRs, and PCBs via dust ingestion were 1.1–24.1, 0.73–20.3, 1.36–23.5, and 0.04–0.93 ng/kg bw/day for adults, and 16.2–352, 10.7–296, 19.9–343, 0.05–0.61, 0.65–13.6 ng/kg bw/day for toddlers, respectively. Residents from Site 5 had the highest exposure (95 percentile levels and high dust ingestion for toddlers) of PBDEs (3920 ng/kg bw/day), NBFRs (3200 ng/kg bw/day), and PFRs (5280 ng/kg bw/day). More attention should be paid to the contamination with NBFRs and PFRs, instead of PCBs, in these e-waste recycling regions, and local public health threat from PBDE alternatives should remain of concern. To the best of our knowledge, this is the first study on human exposure assessment of PFRs at e-waste sites. Keywords: Electronic-waste recycling, Flame retardants, Human exposure, Indoor dust

Requirements for the measurement resolution in the sub-nanometer range have become quite common which includes not only the repeatability or reproducibility but also the absolute measurement accuracy. The freeform lens for wavefront compensating contains some medium spatial frequency terms. The wavefront error of lithographic object lens is very small. One method to reduce the wavefront error of lithographic object lens is to use the freeform lens. The freeform lens for compensation needs more accuracy than the object lens. We can also use freeform lens of sphere or aspheric for compensation. The testing accuracy of sphere and aspheric lens are hard to achieve 1nm. The sphere and aspheric will contain the power term and are hard to find the cat-eye. The ion beam figure system (IBF) is the best polishing machine for nanometer manufacture which will polish the PV of 2um for several weeks even months. Usually we use the PV 200nm lens for compensation. So the freeform for compensation looks like a flat. In this paper we will show the testing experiment of the freeform and the testing problem. The freeform surface is created by 66 Zernike polynomials which are based on the flat lens. The freeform flat is polished by the ion figuring machine of NTG. The environment such as temperature, vibration, humidity is controlled well. The Zygo's interferometer Verifire Ashpere with absolute testing method is used to test the freeform. Position Accuracy is a problem in optical testing and manufacture. The high accuracy testing can’t be determined by one method, we need the different method to compare the result especially these method will contain some defects. The defects of the recently absolute testing method are discussed.

Human biomonitoring (HBM) is a scientific technique that allows us to assess whether and to what extent environmental pollutants enter humans. We review here the current HBM efforts for organophosphate esters, emerging flame retardants, perfluoroalkyl substances, and phthalate esters. Use of some of these chemicals has already been banned or restricted; they are regularly detected in the environment, wildlife, and human matrices. Traditionally, blood and urine collection have been widely used as sampling methods. New non-invasive approaches (e.g., saliva, hair, nails) are emerging as valid alternatives since they offer advantages with respect to sampling, handling, and ethical aspects, while ensuring similar reliability and sensitivity. Nevertheless, the identification of biomarkers of exposure is often difficult because chemicals may be metabolized in the human body. For many of the above-mentioned compounds, the mechanisms of the favorable metabolization pathways have not been unraveled, but research on important metabolites that could be used as biomarkers of exposure is growing. This review summarizes the state of the art regarding human exposure to, (non-invasive) HBM of, and metabolism of major organophosphate esters, emerging flame retardants, perfluoroalkyl substances, and phthalate esters currently detected in the environment.

In Cartesian coordinate system, a flat can be expressed as the sum of even-odd, odd-even, even-even and oddodd functions. In the traditional three-flat even and odd function method, odd-odd function is difficult to obtain. In our paper the odd-odd function can be solved by use the Dove prism which can rotate the optical axis. The odd-odd function can calculate exactly. The even-odd, odd-even, even-even can be solved by rotating the flat 180°like the traditional method. Only five configurations are used to test the flats. The theoretical derivation and analysis are presented.

Large-aperture optical elements are widely employed in high-power laser system, astronomy, and outer-space technology. Sub-aperture stitching is an effective way to extend the lateral and vertical dynamic range of a conventional interferometer. With the aim to provide the accuracy of equipment, this paper simulates the arithmetic to analyze the errors. The Selection of stitching mode and setting of the number of subaperture is given. According to the programmed algorithms simulation stitching is performed for testing the algorithm. In this paper, based on the Matlab we simulate the arithmetic of Sub-aperture stitching. The sub-aperture stitching method can also be used to test the free formed surface. The freeformed surface is created by Zernike polynomials. The accuracy has relationship with the errors of tilting, positioning. Through the stitching the medium spatial frequency of the surface can be tested. The results of errors analysis by means of Matlab are shown that how the tilting and positioning errors to influence the testing accuracy. The analysis of errors can also be used in other interferometer systems.

The projection objectives，used in modern projection lithography, such as deep-ultraviolet lithography (DUVL), or extreme-ultraviolet lithography (EUVL), desire very high-quality optics. It has placed stringent requirements on the accuracy of the interferometers used for optical metrology. Point diffraction interferometry, which generates a spherical reference wave front by pinhole diffraction, has been developed to meet this need. In order to estimate the measurement accuracy, several scalar wave diffraction methods have been used when the diameter of the pinhole is much larger than the wavelength. However, while the diameter keeps decreasing, ultimately to the order of the wavelength, it is obviously not appropriate to calculate in the same way. So, a three-dimensional (3-D) electromagnetic field simulation, based on Finite Element Method (FEM), is set up to study the propagation of the visible- light, 632.8 nm wavelength, through sub- 1000nm diameter pinholes in a chromium membrane. Deviations have been calculated to predict the accuracy, between perfect sphere and the wave front, diffracted by a series of pinholes with different diameters.

BbBSLS and BbBEAS were dissected and reconstituted in Saccharomyces cerevisiae. The intermodular linker is essential for the reconstitution of the separate modules. Module 1 can be swapped between BbBEAS and BbBSLS, while modules 2 and 3 control the product profiles. BbBSLS is a flexible enzyme that also synthesizes beauvericins.