Hyers-Ulam-Rassias Stability of Some Additive Fuzzy Set-Valued Functional Equations with the Fixed Point Alternative

Let Y be a real separable Banach space and let CY,d∞ be the subspace of all normal fuzzy convex and upper semicontinuous fuzzy sets of Y equipped with the supremum metric d∞. In this paper, we introduce several types of additive fuzzy set-valued functional equations in CY,d∞. Using the fixed point technique, we discuss the Hyers-Ulam-Rassias stability of three types additive fuzzy set-valued functional equations, that is, the generalized Cauchy type, the Jensen type, and the Cauchy-Jensen type additive fuzzy set-valued functional equations. Our results can be regarded as important extensions of stability results corresponding to single-valued functional equations and set-valued functional equations, respectively

Transformation of \u3ci\u3eFusarium verticillioides\u3c/i\u3e with a polyketide gene cluster isolated from a fungal endophyte activates the biosynthesis of fusaric acid

A large number of bioactive natural products have been isolated from plant endophytic fungi. However, molecular mechanisms for the biosynthesis of these metabolites have lagged behind because genetic and biochemical studies are difficult to perform within many of the endophytes. In this work, we describe our attempt to express a putative mycoepoxydiene (MED) biosynthetic gene cluster in Fusarium verticillioides, which has a well-developed genetic system for the study fungal polyketide biosynthesis. MED was isolated from Phomopsis sp. A123, a fungal endophyte of the mangrove plant, Kandelia candel. It has several unusual structural features and interesting biological activities. Integration of this Phomopsis gene cluster into the F. verticillioides genome led to the biosynthesis of multiple metabolites. The most highly activated metabolite was isolated and its structure was shown by 1D- and 2D-NMR to be fusaric acid, which is a mycotoxin in Fusarium species and is implicated in fungal pathogenesis. Although fusaric acid was isolated more than 70 years ago, its biosynthetic mechanism remains unclear. These transformants produced 30–35 mg fusaric acid per 100 ml culture. The high level production of fusaric acid will greatly facilitate the genetic and biochemical study of its biosynthetic mechanism. Although we have not detected MED or its analogs from the heterologous host, this work represents the first attempt to express a fungal endophytic gene cluster in a Fusarium species

HSAF-induced antifungal effects in Candida albicans through ROS-mediated apoptosis

Heat-stable antifungal factor (HSAF) belongs to polycyclic tetramate macrolactams (PTMs), which inhibits many fungal pathogens and is effective in inhibiting Candida albicans (C. albicans). In this study, we found that HSAF induced the apoptosis of C. albicans SC5314 through inducing the production of reactive oxygen species (ROS). Nevertheless, we validated the efficacy of HSAF against candidiasis caused by C. albicans in a murine model in vivo,and HSAF significantly improved survival and reduced fungal burden compared to vehicles. A molecular dynamics (MD) simulation was also investigated, revealing the theoretical binding mode of HSAF to the β-tubulin of C. albicans. This study first found PTMs-induced fungal apoptosis through ROS accumulation in C. albicans and its potential as a novel agent for fungicides

Localization Accuracy of Ultrasound-Actuated Needle with Color Doppler Imaging

An ultrasonic needle-actuating device for tissue biopsy and regional anaesthesia offers enhanced needle visibility with color Doppler imaging. However, its specific performance is not yet fully determined. This work investigated the influence on needle visibility of the insertion angle and drive voltage, as well as determined the accuracy and agreement of needle tip localization by comparing color Doppler measurements with paired photographic and B-mode ultrasound measurements. Needle tip accuracy measurements in a gelatin phantom gave a regression trend, where the slope of trend is 0.8808; coefficient of determination (R2) is 0.8877; bias is −0.50 mm; and the 95% limits of agreement are from −1.31 to 0.31 mm when comparing color Doppler with photographic measurements. When comparing the color Doppler with B-mode ultrasound measurements, the slope of the regression trend is 1.0179; R2 is 0.9651; bias is −0.16 mm; and the 95% limits of agreement are from −1.935 to 1.605 mm. The results demonstrate the accuracy of this technique and its potential for application to biopsy and ultrasound guided regional anaesthesia

Iterative Assembly of Two Separate Polyketide Chains by the Same Single-module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

HSAF (1) was isolated from the biocontrol agent Lysobacter enzymogenes (Figure 1).[1-4] This bacterial metabolite belongs to polycyclic tetramate macrolactams (PTM) that are emerging as a new class of natural products with distinct structural features. [5, 6] HSAF exhibits a potent antifungal activity and shows a novel mode of action.[1-4] The HSAF biosynthetic gene cluster contains only a single-module hybrid polyketide synthasenonribosomal peptide synthetase (PKS-NRPS), although the PTM scaffold is apparently derived from two separate hexaketide chains and an ornithine residue.[1-4] This suggests that the same PKS module would act not only iteratively, but also separately, in order to link the two hexaketide chains with the NRPS-activated ornithine to form the characteristic PTM scaffold. Recently, the Gulder group reported heterologous expression of the ikarugamycin (4) biosynthetic gene cluster in E. coli,[7] and the Zhang group reported the enzymatic mechanism for formation of the inner 5-memebered ring and demonstrated the polyketide origin of the ikarugamycin skeleton.[8] Ikarugamycin is a Streptomyces-derived PTM which has a 5,6,5-tricyclic system (Figure 1). Both the Gulder and Zhang groups showed that a three-gene cluster is sufficient for ikarugamycin biosynthesis. Despite the progress, this iterative polyketide biosynthetic mechanism had not been demonstrated using purified PKS and NRPS. In addition, HSAF has a 5,5,6-tricyclic system, and its gene cluster contains at least six genes.[3] Finally, unlike most PTM compounds, HSAF is produced by a Gramnegative bacterium, L. enzymogenes. Here, we report the heterologous production of HSAF analogs in Gram-positive Streptomyces hosts, in which the native PKS have been deleted. We also obtained evidence for the formation of the polyene tetramate intermediate in Streptomyces when only the single-module hybrid PKS-NRPS gene was expressed. Finally, we showed the in vitro production of the polyene tetramate using the individually purified PKS and NRPS. The results provide direct evidence for this iterative polyketide biosynthetic mechanism that is likely general for the PTM-type hybrid polyketide-peptides

Tricarballylic ester formation during biosynthesis of fumonisin mycotoxins in \u3ci\u3eFusarium verticillioides\u3c/i\u3e

Fumonisins are agriculturally important mycotoxins produced by the maize pathogen Fusarium verticillioides. The chemical structure of fumonisins contains two tricarballylic esters, which are rare structural moieties and important for toxicity. The mechanism for the tricarballylic ester formation is not well understood. FUM7 gene of F. verticillioides was predicted to encode a dehydrogenase/reductase, and when it was deleted, the mutant produced tetradehydro fumonisins (DH4–FB). MS and NMR analysis of DH4–FB1 indicated that the esters consist of aconitate with a 3′-alkene function, rather than a 2′-alkene function. Interestingly, the purified DH4–FB1 eventually yielded three chromatographic peaks in HPLC. However, MS revealed that the metabolites of the three peaks all had the same mass as the initial single-peak DH4–FB1. The results suggest that DH4–FB1 can undergo spontaneous isomerization, probably including both cis–trans stereoisomerization and 3′- to 2′-ene regioisomerization. In addition, when FUM7 was expressed in Escherichia coli and the resulting enzyme, Fum7p, was incubated with DH4–FB, no fumonisin with typical tricarballylic esters was formed. Instead, new fumonisin analogs that probably contained isocitrate and/or oxalosuccinate esters were formed, which reveals new insight into fumonisin biosynthesis. Together, the data provided both genetic and biochemical evidence for the mechanism of tricarballylic ester formation in fumonisin biosynthesis

Tricarballylic ester formation during biosynthesis of fumonisin mycotoxins in \u3ci\u3eFusarium verticillioides\u3c/i\u3e

Fumonisins are agriculturally important mycotoxins produced by the maize pathogen Fusarium verticillioides. The chemical structure of fumonisins contains two tricarballylic esters, which are rare structural moieties and important for toxicity. The mechanism for the tricarballylic ester formation is not well understood. FUM7 gene of F. verticillioides was predicted to encode a dehydrogenase/reductase, and when it was deleted, the mutant produced tetradehydro fumonisins (DH4–FB). MS and NMR analysis of DH4–FB1 indicated that the esters consist of aconitate with a 3′-alkene function, rather than a 2′-alkene function. Interestingly, the purified DH4–FB1 eventually yielded three chromatographic peaks in HPLC. However, MS revealed that the metabolites of the three peaks all had the same mass as the initial single-peak DH4–FB1. The results suggest that DH4–FB1 can undergo spontaneous isomerization, probably including both cis–trans stereoisomerization and 3′- to 2′-ene regioisomerization. In addition, when FUM7 was expressed in Escherichia coli and the resulting enzyme, Fum7p, was incubated with DH4–FB, no fumonisin with typical tricarballylic esters was formed. Instead, new fumonisin analogs that probably contained isocitrate and/or oxalosuccinate esters were formed, which reveals new insight into fumonisin biosynthesis. Together, the data provided both genetic and biochemical evidence for the mechanism of tricarballylic ester formation in fumonisin biosynthesis

Marek's disease virus-encoded miR-155 ortholog critical for the induction of lymphomas is not essential for the proliferation of transformed cell lines

MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.IMPORTANCE Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype

Identification and Characterization of the Anti-Methicillin-Resistant \u3ci\u3eStaphylococcus aureus\u3c/i\u3e WAP-8294A2 Biosynthetic Gene Cluster from \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e OH11

Lysobactor enzymogenes strain OH11 is an emerging biological control agent of fungal and bacterial diseases. We recently completed its genome sequence and found it contains a large number of gene clusters putatively responsible for the biosynthesis of nonribosomal peptides and polyketides, including the previously identified antifungal dihydromaltophilin (HSAF). One of the gene clusters contains two huge open reading frames, together encoding 12 modules of nonribosomal peptide synthetases (NRPS). Gene disruption of one of the NRPS led to the disappearance of a metabolite produced in the wild type and the elimination of its antibacterial activity. The metabolite and antibacterial activity were also affected by the disruption of some of the flanking genes. We subsequently isolated this metabolite and subjected it to spectroscopic analysis. The mass spectrometry and nuclear magnetic resonance data showed that its chemical structure is identical to WAP-8294A2, a cyclic lipodepsipeptide with potent antimethicillin-resistant Staphylococcus aureus (MRSA) activity and currently in phase I/II clinical trials. The WAP- 8294A2 biosynthetic genes had not been described previously. So far, the Gram-positive Streptomyces have been the primary source of anti-infectives. Lysobacter are Gram-negative soil/water bacteria that are genetically amendable and have not been well exploited. The WAP-8294A2 synthetase represents one of the largest NRPS complexes, consisting of 45 functional domains. The identification of these genes sets the foundation for the study of the WAP-8294A2 biosynthetic mechanism and opens the door for producing new anti-MRSA antibiotics through biosynthetic engineering in this new source of Lysobacter