A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 T cells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Chemometrics and genome mining reveal an unprecedented family of sugar acid-containing fungal nonribosomal cyclodepsipeptides.

Xylomyrocins, a unique group of nonribosomal peptide secondary metabolites, were discovered in Paramyrothecium and Colletotrichum spp. fungi by employing a combination of high-resolution tandem mass spectrometry (HRMS/MS)-based chemometrics, comparative genome mining, gene disruption, stable isotope feeding, and chemical complementation techniques. These polyol cyclodepsipeptides all feature an unprecedented d-xylonic acid moiety as part of their macrocyclic scaffold. This biosynthon is derived from d-xylose supplied by xylooligosaccharide catabolic enzymes encoded in the xylomyrocin biosynthetic gene cluster, revealing a novel link between carbohydrate catabolism and nonribosomal peptide biosynthesis. Xylomyrocins from different fungal isolates differ in the number and nature of their amino acid building blocks that are nevertheless incorporated by orthologous nonribosomal peptide synthetase (NRPS) enzymes. Another source of structural diversity is the variable choice of the nucleophile for intramolecular macrocyclic ester formation during xylomyrocin chain termination. This nucleophile is selected from the multiple available alcohol functionalities of the polyol moiety, revealing a surprising polyspecificity for the NRPS terminal condensation domain. Some xylomyrocin congeners also feature N-methylated amino acid residues in positions where the corresponding NRPS modules lack N-methyltransferase (M) domains, providing a rare example of promiscuous methylation in the context of an NRPS with an otherwise canonical, collinear biosynthetic program.

Dabigatran-related serious medication errors: an analysis using data from VigiBase.

OBJECTIVE: To analyze the serious medication errors (MEs) on dabigatran, and their related factors, in order to avoid or reduce the occurrence of adverse events. METHODS: Serious MEs related to dabigatran were extracted from the WHO global database of reported potential side effects of medicinal products (VigiBase) by using "Medication errors and other product use errors and issues" High Level Group Term (HLGT) of the international Medical Dictionary for Regulatory Activities (MedDRA). Well-documented reports, vigiGrade completeness score ≥ 0.80, or with an informative narrative were analyzed with a focus on the clinical features of the cases. The PCNE Classification for drug-related problems (DRP) was used to classify medication errors in our analysis of cases. RESULTS: Until January 26, 2020, there were 453 cases with serious MEs related to dabigatran in VigiBase, and 113 were well-documented. Among these, 69 patients (61%) were hospitalized or had prolonged hospitalization, 16 (14%) had life-threatening events, and 12 (11%) died. The MEs occurred in the prescription phase in 77 cases, in administration in 35, and at the dispensing stage in one case. The MEs in prescription were related to a drug selection error in 44 cases (24 concerning contraindications and 20 drug interactions) and to dose error in 33 cases (17 with excessive dose; eight with insufficient frequency; four had an incorrect time; in three, the dose was too low; and in one, too frequent). The MEs in administration were medical-staff-related errors in five cases (three with wrong administration route, one administration omission, and one overdose), patient-related errors in 28 (14 insufficient dose or no administration, seven improper drug storage, four wrong administration method, and three over prescribed dose), and other errors in two (without efficacy monitoring). The dispensing error of a wrong drug strength occurred in a pharmacy. The main adverse events in the 113 patients were haemorrhage in 57 cases (50%) and ischemia in 29 cases (26%). CONCLUSION: Based on the analysis of reports in VigiBase, serious MEs related to dabigatran mainly occurred during prescription and administration. Although the incidence of MEs with clinical consequences in the use of dabigatran cannot be determined, attention should be paid to selection of the appropriate dose to a right patient in the prescription, and to patient compliance and storage in drug administration. The patient harm mainly manifested itself as bleeding or ischemia including fatal outcome in rare patients.

Re-Engineering Fungal Nonribosomal Peptide Synthetases by Module Dissection and Duplicated Thiolation Domains.

Unnatural product (uNP) nonribosomal peptides promise to be a valuable source of pharmacophores for drug discovery. However, the extremely large size and complexity of the nonribosomal peptide synthetase (NRPS) enzymes pose formidable challenges to the production of such uNPs by combinatorial biosynthesis and synthetic biology. Here we report a new NRPS dissection strategy that facilitates the engineering and heterologous production of these NRPSs. This strategy divides NRPSs into "splitting units", each forming an enzyme subunit that contains catalytically independent modules. Functional collaboration between the subunits is then facilitated by artificially duplicating, at the N-terminus of the downstream subunit, the linker - thiolation domain - linker fragment that is resident at the C-terminus of the upstream subunit. Using the suggested split site that follows a conserved motif in the linker connecting the adenylation and the thiolation domains allows cognate or chimeric splitting unit pairs to achieve productivities that match, and in many cases surpass those of hybrid chimeric enzymes, and even those of intact NRPSs, upon production in a heterologous chassis. Our strategy provides facile options for the rational engineering of fungal NRPSs and for the combinatorial reprogramming of nonribosomal peptide production.

Five Porous Complexes Constructed from a Racemic Ligand: Synthesis, Chiral Self-Assembly, Iodine Adsorption, and Desorption Properties.

Herein, a chiral bispyridyl ligand (L) was designed and synthesized using a Schiff base condensation reaction, followed by a 1,3-H shift. Five complexes, [Zn2L2(OAc)4] (1), {[CdLCl2(DMF)]·4H2O}n (2), [Cd2L2I4]·4H2O (3), {[CdL2(H2O)2](NO3)2·2CH3OH}n (4), and [Hg2L2Cl4]·2DMF (5), were synthesized and characterized upon its reaction with Zn(II), Cd(II), or Hg(II) ions, respectively. X-ray crystallography shows that the organic compound exists as a racemic ligand with equal amounts of its R- and S-isomers, and all of the synthesized complexes exhibit heterochiral self-assembly via a chiral self-discrimination process. Complexes 1, 3, and 5 exist as centrosymmetric binuclear metallamacrocycles, while complexes 2 and 4 exist as 1D looped-chain coordination polymers. Inspired by the assembled structures of the five complexes, I2 adsorption/desorption measurements for the complexes were carried out. The results show that complexes 1 and 5 exhibit good adsorption capacities toward I2 in n-hexane and in water, respectively.

Coordination-Driven Heterochiral Self-Assembly: Construction of Cd(II) Coordination Polymers with Sorption Behaviors for Iodine and Dyes.

A racemic bispyridyl ligand (L) was synthesized via a Schiff base condensation reaction. Four Cd(II) complexes, {[CdL2Cl2]·2DMF}n (1), [CdLI2]n (2), {[CdL2Br2]·4H2O}n (3), and {[CdL2(H2O)2](NO3)2·2CH3OH·8H2O}n (4), were synthesized and further characterized based on this ligand. Single-crystal structures show that the coordination-driven assembly of the bispyridyl ligand with Cd(II) salts bearing different counteranions can lead to multidimensional coordination polymers via a heterochiral self-discrimination process. Complex 1 exists as a one-dimensional (1D) looped chain polymer, and complex 2 exists as a 1D zigzag chain polymer. Complex 3 is a 2D grid coordination polymer, and complex 4 exists as a 3D framework polymer. Furthermore, the iodine sorption capacities of the four complexes were investigated in the solution of n-hexane and water as well as in the iodine steam. The dye sorption behaviors were investigated in water, which showed that complex 2 exhibited good adsorption for crystal violet (CV), while complex 4 had good adsorption capability toward direct yellow 4 (DY).

Mass Spectrometry-Guided Discovery of Multi-N-Methylated Cyclodecapeptides Auyuittuqamides E-H from Sesquicillium sp. QL0466.

Mass spectrometry-based dereplication and prioritization led to the discovery of four multi-N-methylated cyclodecapeptides, auyuittuqamides E-H (1-4), from a soil-derived Sesquicillium sp. The planar structures of these compounds were elucidated based on analysis of HRESIMS and NMR data. Absolute configurations of the chiral amino acid residues were assigned by a combination of the advanced Marfey's method, chiral-phase LC-MS analysis, and J-based configuration analysis, revealing that 1-4 contain both d- and l-isomers of N-methylleucine (MeLeu). Differentiation of d- and l-MeLeu in the sequence was achieved by advanced Marfey's analysis of the diagnostic peptide fragments generated from partial hydrolysis of 1. Bioinformatic analysis identified a putative biosynthetic gene cluster (auy) for auyuittuqamides E-H, and a plausible biosynthetic pathway was proposed. These newly identified fungal cyclodecapeptides (1-4) displayed in vitro growth inhibitory activity against vancomycin-resistant Enterococcus faecium with MIC values of 8 µg/mL.

Fatal adverse events of rivaroxaban combined with aspirin: an analysis using data from VigiBase.

PURPOSE: The aim of this study was to analyze the clinical characteristics of fatal adverse events (AEs) of rivaroxaban combined with aspirin and to underline the importance of the rational use of drugs. METHODS: The WHO global database of reported potential side effects of medicinal products (VigiBase) was searched for fatal AEs in the combined use of rivaroxaban and aspirin, and the clinical characteristics of those cases with sufficient information (vigiGrade completeness score ≥ 0.80) were analyzed. RESULTS: By January 19, 2020, 2309 fatal adverse event reports of rivaroxaban combined with aspirin from 21 countries were entered in VigiBase. One hundred and twenty cases contained further information, of which 42 were female (35%) and 78 were male (65%). The median age was 75 (range 34 to 93) years, and 109 cases (91%) were elderly patients (≥ 65 years). The AEs listed in the fatal case reports included bleeding in 114 cases (mainly intracranial hemorrhage and gastrointestinal hemorrhage, 59 and 46 respectively, accounting for 88%) and ischemic events in six cases (ischemic stroke in three, acute myocardial infarction in two, myocardial infarction combined with acute liver failure in one). Among the patients with bleeding events, 108 (95%) had existing risk factors for bleeding or for interacting with aspirin or rivaroxaban. These may be divided into the following: diseases (hypertension, renal impairment, history of stroke, peptic ulcer, or previous bleeding), drugs (high dose aspirin, antiplatelet drugs, anticoagulants, P-gp inhibitors/CYP3A4 inhibitors, non-steroidal anti-inflammatory drugs, steroids, and selective serotonin reuptake inhibitors), or other factors (e.g., elderly, low body weight, or excessive intake of ginger, fish oil, or alcohol). There were 45 cases with two or more of these risk factors in addition to rivaroxaban and aspirin. Patients with ischemic events are often in very high-risk groups of atherosclerotic cardiovascular disease (ASCVD) or self-discontinuation of treated drugs. Medication errors occurred in 24 patients (20%): excessive treatment in 17 cases, contraindication in three, frequency error in two, excessive treatment combined with contraindication in one, and self-discontinuation in one. CONCLUSIONS: Fatal AEs related to rivaroxaban combined with aspirin, including bleeding and ischemic events, have been reported mostly in the elderly, and sometimes involved medication errors. The fatal AEs mainly manifested as serious bleeding, and most of them occurred in patients with concurrent multiple risk factors. Monitoring coagulation during rivaroxaban treatment is recommended in very high-risk ASCVD populations, and attention should be paid to prevention of medication errors.

Downregulation of lncRNA ZFAS1 and upregulation of microRNA-129 repress endocrine disturbance, increase proliferation and inhibit apoptosis of ovarian granulosa cells in polycystic ovarian syndrome by downregulating HMGB1.

OBJECTIVE: The objective was to find the role of long-non-coding RNA zinc finger antisense 1 (lncRNA ZFAS1)/microRNA (miR)-129/high-mobility group box protein 1 (HMGB1) axis in polycystic ovary syndrome (PCOS). METHODS: Ovarian granulosa cells from non-PCOS patients and PCOS patients were collected, and HMGB1, miR-129 and lncRNA ZFAS1 expression were detected. Ovarian granulosa cells were transfected with si-ZFAS1 or miR-129 mimics to verify their roles in P4 and E2 secretion, and the biological functions of ovarian granulosa cells. RESULTS: LncRNA ZFAS1 and HMGB1 were elevated, while miR-129 was down-regulated in ovarian granulosa cells of PCOS patients. Down-regulated lncRNA ZFAS1 or overexpressed miR-129 could decrease HMGB1 expression, increase P4 and E2 secretion, promote proliferation activity while inhibit apoptosis of ovarian granulosa cells in PCOS. CONCLUSION: LncRNA ZFAS1 could bind to miR-129 to promote HMGB1 expression, thereby affecting the endocrine disturbance, proliferation and apoptosis of ovarian granulosa cells in PCOS.

The regulation of BbLaeA on the production of beauvericin and bassiatin in Beauveria bassiana.

Beauvericin and bassiatin are two valuable compounds with various bioactivities biosynthesized by the supposedly same nonribosomal peptide synthetase BbBEAS in entomopathogenic fungus Beauveria bassiana. To evaluate the regulatory effect of global regulator LaeA on their production, we constructed BbLaeA gene deletion and overexpression mutants, respectively. Deletion of BbLaeA resulted in a decrease of the beauvericin titer, while overexpression of BbLaeA increased its production by 1-2.26 times. No bassiatin could be detected in ΔBbLaeA and wild type strain of B. bassiana, but 4.26-5.10 µg/mL bassiatin was produced in OE::BbLaeA. Furthermore, additional metabolites with increased production in OE::BbLaeA were isolated and identified as primary metabolites. Among them, 4-hydroxyphenylacetic acid showed antibacterial bioactivity against Ralstonia solanacearum. These results indicated that BbLaeA positively regulates the production of beauvericin, bassiatin and various bioactive primary metabolites.

Intrinsic and Extrinsic Programming of Product Chain Length and Release Mode in Fungal Collaborating Iterative Polyketide Synthases.

Combinatorial biosynthesis with fungal polyketide synthases (PKSs) promises to produce unprecedented bioactive "unnatural" natural products (uNPs) for drug discovery. Genome mining of the dothideomycete Rhytidhysteron rufulum uncovered a collaborating highly reducing PKS (hrPKS)-nonreducing PKS (nrPKS) pair. These enzymes produce trace amounts of rare S-type benzenediol macrolactone congeners with a phenylacetate core in a heterologous host. However, subunit shuffling and domain swaps with voucher enzymes demonstrated that all PKS domains are highly productive. This contradiction led us to reveal novel programming layers exerted by the starter unit acyltransferase (SAT) and the thioesterase (TE) domains on the PKS system. First, macrocyclic vs linear product formation is dictated by the intrinsic biosynthetic program of the TE domain. Next, the chain length of the hrPKS product is strongly influenced in trans by the off-loading preferences of the nrPKS SAT domain. Last, TE domains are size-selective filters that facilitate or obstruct product formation from certain priming units. Thus, the intrinsic programs of the SAT and TE domains are both part of the extrinsic program of the hrPKS subunit and modulate the observable metaprogram of the whole PKS system. Reconstruction of SAT and TE phylogenies suggests that these domains travel different evolutionary trajectories, with the resulting divergence creating potential conflicts in the PKS metaprogram. Such conflicts often emerge in chimeric PKSs created by combinatorial biosynthesis, reducing biosynthetic efficiency or even incapacitating the system. Understanding the points of failure for such engineered biocatalysts is pivotal to advance the biosynthetic production of uNPs.

Secondary metabolites from hypocrealean entomopathogenic fungi: genomics as a tool to elucidate the encoded parvome.

Covering: 2014 up to the third quarter of 2019 Hypocrealean entomopathogenic fungi (HEF) produce a large variety of secondary metabolites (SMs) that are prominent virulence factors or mediate various interactions in the native niches of these organisms. Many of these SMs show insecticidal, immune system modulatory, antimicrobial, cytotoxic and other bioactivities of clinical or agricultural significance. Recent advances in whole genome sequencing technologies and bioinformatics have revealed many biosynthetic gene clusters (BGCs) potentially involved in SM production in HEF. Some of these BGCs are now well characterized, with the structures of the cognate product congeners elucidated, and the proposed biosynthetic functions of key enzymes validated. However, the vast majority of HEF BGCs are still not linked to SM products ("orphan" BGCs), including many clusters that are not expressed (silent) under routine laboratory conditions. Thus, investigations into the encoded parvome (the secondary metabolome predicted from the genome) of HEF allows the discovery of BGCs for known SMs; uncovers novel metabolites based on the BGCs; and catalogues the predicted SM biosynthetic potential of these fungi. Herein, we summarize new developments of the field, and survey the polyketide, nonribosomal peptide, terpenoid and hybrid SM BGCs encoded in the currently available 40 HEF genome sequences. Studying the encoded parvome of HEF will increase our understanding of the multifaceted roles that SMs play in biotic and abiotic interactions and will also reveal biologically active SMs that can be exploited for the discovery of human and veterinary drugs or crop protection agents.

Discovery of a new antifungal lipopeptaibol from Purpureocillium lilacinum using MALDI-TOF-IMS.

Fungi are considered to be rich in biologically active natural products for agricultural and medicinal purposes. The discovery and accurate identification of the bioactive fungal natural products is important for their efficient utilization. During the course of our continuing search for the new natural products from the fungal agents, we found the well-known bio-control fungus Purpureocillium lilacinum showed in vitro activity against Botrytis cinerea, an airborne plant pathogenic fungus causing gray mold disease in many vegetables and fruits. The co-culture of two fungi on agar plate showed that P. lilacinum inhibited the growth of B. cinerea which means P. lilacinum has potential to produce some bioactive secondary metabolites against B. cinerea. In this study, we applied matrix-assisted laser desorption ionization-time of flight mass spectrometry imaging mass spectrometry (MALDI-TOF-IMS), as a fast identification tool, for the discovery of a new antifungal lipopeptaibol (leucinostatin Z) from P. lilacinum against B. cinerea. The planar structure of leucinostatin Z was further established by using the LC-HRESI-MS-MS analysis. MALDI-TOF-IMS is becoming a new approach that allows us to observe the bioactive natural products directly on growth media between the colonies of two fungi, which is faster and more effective than the traditional techniques to discover new bioactive compounds in fungi.

SWEDEGENE-a Swedish nation-wide DNA sample collection for pharmacogenomic studies of serious adverse drug reactions.

SWEDEGENE is a Swedish nation-wide sample collection established to facilitate studies of clinical and genetic risk factors for adverse drug reactions (ADRs). Most cases are recruited among patients reported to the ADR registry at the Swedish Medical Products Agency by health-care professionals. Clinical data are collected both from medical and laboratory records and through interviews using standardized questionnaires. Genome-wide scans and whole-genome sequencing are done, and association studies are conducted using mainly controls from the Swedish TwinGene biobank with data on diagnoses and prescribed drugs. SWEDEGENE was established in 2008 and currently contains DNA and information from about 2550 adults who have experienced specific ADRs, and from 580 drug exposed controls. Results from genome-wide association studies have now been published, and data from whole-genome sequencing are being analyzed. SWEDEGENE has the potential to offer a new means of developing individualized and safe drug therapy through patient pre-treatment screening.

Genome-wide association study of angioedema induced by angiotensin-converting enzyme inhibitor and angiotensin receptor blocker treatment.

Angioedema in the mouth or upper airways is a feared adverse reaction to angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blocker (ARB) treatment, which is used for hypertension, heart failure and diabetes complications. This candidate gene and genome-wide association study aimed to identify genetic variants predisposing to angioedema induced by these drugs. The discovery cohort consisted of 173 cases and 4890 controls recruited in Sweden. In the candidate gene analysis, ETV6, BDKRB2, MME, and PRKCQ were nominally associated with angioedema (p < 0.05), but did not pass Bonferroni correction for multiple testing (p < 2.89 × 10-5). In the genome-wide analysis, intronic variants in the calcium-activated potassium channel subunit alpha-1 (KCNMA1) gene on chromosome 10 were significantly associated with angioedema (p < 5 × 10-8). Whilst the top KCNMA1 hit was not significant in the replication cohort (413 cases and 599 ACEi-exposed controls from the US and Northern Europe), a meta-analysis of the replication and discovery cohorts (in total 586 cases and 1944 ACEi-exposed controls) revealed that each variant allele increased the odds of experiencing angioedema 1.62 times (95% confidence interval 1.05-2.50, p = 0.030). Associated KCNMA1 variants are not known to be functional, but are in linkage disequilibrium with variants in transcription factor binding sites active in relevant tissues. In summary, our data suggest that common variation in KCNMA1 is associated with risk of angioedema induced by ACEi or ARB treatment. Future whole exome or genome sequencing studies will show whether rare variants in KCNMA1 or other genes contribute to the risk of ACEi- and ARB-induced angioedema.

Apc.LaeA and Apc.VeA of the velvet complex govern secondary metabolism and morphological development in the echinocandin-producing fungus Aspergillus pachycristatus.

The impact of the global secondary metabolite regulators LaeA and VeA on echinocandin B production and morphological development was evaluated in the industrial production strain Aspergillus pachycristatus NRRL 11440. Other representative secondary metabolites were examined as well to determine if the velvet complex functions as in A. nidulans and other species of fungi. Genetic methods used for gene manipulations in A. nidulans were applied to A. pachycristatus. Separate deletions of genes Apc.laeA and Apc.veA resulted in similar yet differing phenotypes in strain NRRL 11440. Disruption of Apc.laeA and Apc.veA significantly reduced, but did not eliminate, the production of echinocandin B. Similar to what has been observed in A. nidulans, the production of sterigmatocystin was nearly eliminated in both mutants. Quantitative reverse transcription PCR analyses confirmed that selected genes of both the echinocandin B and sterigmatocystin gene clusters were down-regulated in both mutant types. The two mutants differed with respect to growth of aerial hyphae, pigmentation, development of conidiophores, conidial germination rate, and ascospore maturation. Further functional annotation of key regulatory genes in A. pachycristatus and related Aspergillus species will improve our understanding of regulation of echinocandin production and co-produced metabolites.

Genomics-driven discovery of a novel self-resistance mechanism in the echinocandin-producing fungus Pezicula radicicola.

The echinocandins are antifungal lipopeptides targeting fungi via noncompetitive inhibition of the ß-1,3-d-glucan synthase FKS1 subunit. A novel echinocandin resistance mechanism involving an auxiliary copy of FKS1 in echinocandin-producing fungus Pezicula radicicola NRRL 12192 was discovered. We sequenced the genome of NRRL 12192 and predicted two FKS1-encoding genes (prfks1n and prfks1a), rather than a single FKS1 gene typical of filamentous ascomycetes. The prfks1a gene sits immediately adjacent to an echinocandin (sporiofungin) gene cluster, which was confirmed by disruption of prnrps4 and abolishment of sporiofungin production. Disruption of prfks1a dramatically increased the strain's sensitivity to exogenous echinocandins. In the absence of echinocandins, transcription levels of prfks1a relative to ß-tubulin in the wild type and in Δprnrps4 stains were similar. Moreover, prfks1a is consistently transcribed at low levels and is upregulated in the presence of exogenous echinocandin, but not during growth conditions promoting endogenous production of sporiofungin. Therefore, we conclude that prfks1a is primarily responsible for protecting the fungus against extracellular echinocandin toxicity. The presence of unclustered auxiliary copies of FKS1 with high similarity to prfks1a in two other echinocandin-producing strains suggests that this previously unrecognized resistance mechanism may be common in echinocandin-producing fungi of the family Dermataceae of the class Leotiomycetes.

Enfumafungin synthase represents a novel lineage of fungal triterpene cyclases.

Enfumafungin is a glycosylated fernene-type triterpenoid produced by the fungus Hormonema carpetanum. Its potent antifungal activity, mediated by its interaction with ß-1,3-glucan synthase and the fungal cell wall, has led to its development into the semi-synthetic clinical candidate, ibrexafungerp (=SCY-078). We report on the preliminary identification of the enfumafungin biosynthetic gene cluster (BGC) based on genome sequencing, phylogenetic reconstruction, gene disruption, and cDNA sequencing studies. Enfumafungin synthase (efuA) consists of a terpene cyclase domain (TC) fused to a glycosyltransferase (GT) domain and thus represents a novel multifunctional enzyme. Moreover, the TC domain bears a phylogenetic relationship to bacterial squalene-hopene cyclases (SHC) and includes a typical DXDD motif within the active centre suggesting that efuA evolved from SHCs. Phylogenetic reconstruction of the GT domain indicated that this portion of the fusion gene originated from fungal sterol GTs. Eleven genes flanking efuA are putatively involved in the biosynthesis, regulation, transport and self-resistance of enfumafungin and include an acetyltransferase, three P450 monooxygenases, a dehydrogenase, a desaturase and a reductase. A hypothetical scheme for enfumafungin assembly is proposed in which the E-ring is oxidatively cleaved to yield the four-ring system of enfumafungin. EfuA represents the first member of a widespread lineage of fungal SHCs.

Excavating differentially expressed antimicrobial peptides from transcriptome of Larimichthys crocea liver in response to Cryptocaryon irritans.

Larimichthys crocea, the special marine economy fish, owns the largest annual yield for a single species in China. One of the most significant factors affecting large yellow croaker culture is the diseases, especially the threat of marine white spot disease which caused by a protozoan Cryptocaryon irritans. Antimicrobial peptides (AMPs) have been demonstrated to be active against bacterium, fungi and parasites, showing their potential usefulness in aquaculture as substitutes for antibiotics. Many researches have been carried out about the AMPs concentrating on the activity resist on C. irritans, and piscidin-like of L. crocea owning widely antibacterial spectrum and strong activity against C. irritans was screened in our team. In the paper, taking advantage of the large yellow croaker hepatic comparison transcriptome in response to C. irritans at 3d post infection, seven kinds of AMPs have been excavated from the differently expressed genes, including LEAP2 like, LEAP-2A, hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type 4 and bactericidal permeability increasing protein (BPI). Hepcidin, hepcidin-like, piscidin-5-like, piscidin-5-like type4 and BPI were up-regulated to protect large yellow croaker from being damaged by C. irritans infection; while LEAP2 like and LEAP-2A were down-regulated, they might be as a negative-feedback regulation factor or some other regulatory mechanisms to adjust the immune response in the process of C. irritans infection. The differential expression changes were verified with quantitative real-time PCR (qRT-PCR) to illustrate the reliability of the sequenced data. Hearteningly, piscidin-5-like type 4 was a novel type which was high similar to other piscidin-5-like types. Interestingly, the infection may well cause alternative splicing of LEAP-2A mRNA, which was a surprised phenomenon and finding after C. irritans infection, but more further study was needed to be conducted. Therefore, the data showed that these AMPs were involved in the immune response to the C. irritans infection. In all, these results implied that the immune response of AMPs to C. irritans infection was a complex and sophisticated regulatory process.

Nonradiative dynamics determined by charge transfer induced hydrogen bonding: a combined femtosecond time-resolved fluorescence and density functional theoretical study of methyl dimethylaminobenzoate in water.

As a case study of the interplay and the consequence of the interplay between intramolecular charge transfer (ICT) and intermolecular hydrogen (H)-bonding, a combined femtosecond time-resolved fluorescence (fs-TRF) and density functional theoretical (DFT) and time-dependent DFT (TDDFT) study has been conducted on methyl dimethylaminobenzoate (MDMABA) largely in a water solvent. Direct observation of the broadband spectra, anisotropy, and kinetic decays of fs-TRF from photo-excited MDMABA revealed a rapid ICT reaction occurring with a time constant of ∼0.7 ps from an initial locally excited (LE) state identified to have the Laππ* character; this produced a weakly emissive ICT state featuring radiative rate constant decreased by more than two orders of magnitude. The fluorescence of the ICT state is strongly quenched exhibiting a decay time of ∼49.7 ps, unusually faster than the nanosecond range lifetime in a polar aprotic solvent when intersystem crossing (ISC) is the major deactivation channel. This, according to the study of the solvent kinetic isotope effect, is identified to originate from an instantly enhanced strong solute-solvent H-bonding induced by the ICT reaction which allows elimination of the ISC, and enables the nonradiative decay to proceed almost entirely through the otherwise inaccessible internal conversion from the ICT state. The enhancement of H-bonding is verified by the calculation which presents theoretical evidence for not only the binding site and binding energy of the H-bonding configuration but also the electronic and structural characterization, lending support to the twisted ICT (TICT) description of the photo-excited MDMABA. This study contributes a prominent example for the extraordinary ability of water and a decisive role of ICT promoted H-bonding in offering a highly effective molecular mechanism for rapid elimination of the electronic excitation energy. The results contain an important insight for the in-depth understanding of the excited state H-bonding dynamics, and also have significant implication for clarifying the "sunscreen controversy" of the DMABA type of UVB sunscreen molecule.