Generation of Multiple Bioactive Macrolides by Hybrid Modular Polyketide Synthases in Streptomyces venezuelae

AbstractThe plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase, leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products

Hearing Abilities at Ultra-High Frequency in Patients with Tinnitus

ObjectivesTo compare tinnitus patients who have normal hearing between 250 Hz and 8 kHz with normal controls with regard to the ability of each group to hear extended high-frequency pure tone thresholds.MethodsWe enrolled 18 tinnitus patients, each of whom had a threshold of HL <25 dB and threshold differences of <10 dB between ears at frequencies of 250 and 500 Hz and 1, 2, 4, and 8 kHz. We also enrolled age- and gender-matched normal volunteers (10 ears), for each patient. Extended high frequency pure tone audiometry was performed, and the mean hearing thresholds at 10, 12, 14, and 16 kHz of each tinnitus ear were compared with those of the 10 age- and sex-matched normal ears.ResultsOf the 18 patients with tinnitus, 12 had significantly increased hearing thresholds at more than one of the four high frequencies, compared with the normal group. When we assessed results according to frequency, we found that 8 patients had decreased hearing ability at 10 kHz, 10 at 12 kHz, 8 at 14 kHz, and 4 at 16 kHz.ConclusionSome patients with tinnitus who have normal hearing below 8 kHz have decreased hearing ability at extended high-frequencies. Thus, the proportion of patients with tinnitus who have normal hearing over the entire audible range is smaller than in previous reports

Engineered biosynthesis of milbemycins in the avermectin high-producing strain Streptomyces avermitilis

Additional file 3 : Figure S2. HPLC analysis of milbemycins produced from S. avermitilis mutant strains and authentic standard milbemycins

The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2)

Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (∼21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development

Chemoenzymatic Synthesis of Glycosylated Macrolactam Analogues of the Macrolide Antibiotic YC‐17

YC‐17 is a 12‐membered ring macrolide antibiotic produced from Streptomyces venezuelae ATCC 15439 and is composed of the polyketide macrolactone 10‐deoxymethynolide appended with D‐desosamine. In order to develop structurally diverse macrolactam analogues of YC‐17 with improved therapeutic potential, a combined approach involving chemical synthesis and engineered cell‐based biotransformation was employed. Eight new antibacterial macrolactam analogues of YC‐17 were generated by supplying a novel chemically synthesized macrolactam aglycone to S. venezuelae mutants harboring plasmids capable of synthesizing several unnatural sugars for subsequent glycosylation. Some YC‐17 macrolactam analogues were active against erythromycin‐resistant bacterial pathogens and displayed improved metabolic stability in vitro. The enhanced therapeutic potential demonstrated by these glycosylated macrolactam analogues reveals the unique potential of chemoenzymatic synthesis in antibiotic drug discovery and development.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/113147/1/adsc_201500250_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113147/2/2697_ftp.pd

The Value of Measuring Eustachian Tube Aeration on Temporal Bone CT in Patients with Chronic Otitis Media

ObjectivesTo evaluate that the cross-sectional area of the air space in the Eustachian tube (ET) on computed tomography (CT) images could be useful for predicting the postoperative aeration of the middle ear.MethodsThe patient group consisted of 80 patients (80 ears) with chronic otitis media and who underwent middle ear surgery from 2006-2007 and who were followed up for more than 1 yr. The control group consisted of 100 ears of 50 individuals with normal tympanic membranes and who underwent CT for other causes (such as tinnitus or hearing loss). The largest cross-sectional areas of the aerated ET were measured on the coronal images of the temporal bone CT by a single otologist using the computer-based "Region of Interest" picture archiving and communications system. The patient group was divided into two subgroups, 1) those with good postoperative aeration and 2) those with poor postoperative aeration. The largest cross-sectional areas of the aerated ETs were compared between the patients and the controls, and between the patients with good aeration and the patients with poor aeration.ResultsThe mean cross-sectional areas significantly differed between the patient group and the control group, and between the good and poor aeration subgroups (P<0.05 each). The mean area of the poor aeration subgroup was smaller than that of the control group (P<0.05), but the mean area of the good aeration subgroup did not significantly differ from that of the controls.ConclusionThe cross-sectional area of the aerated ET, as measured on the preoperative coronal images of temporal bone CT scans, may be useful for predicting the postoperative condition of the tympanic cavity

A nanohybrid system for taste masking of sildenafil

A nanohybrid was prepared with an inorganic clay material, montmorillonite (MMT), for taste masking of sildenafil (SDN). To further improve the taste-masking efficiency and enhance the drug-release rate, we coated the nanohybrid of SDN–MMT with a basic polymer, polyvinylacetal diethylaminoacetate (AEA). Powder X-ray diffraction and Fourier transform infrared experiments showed that SDN was successfully intercalated into the interlayer space of MMT. The AEA-coated SDN–MMT nanohybrid showed drug release was much suppressed at neutral pH (release rate, 4.70 ± 0.53%), suggesting a potential for drug taste masking at the buccal cavity. We also performed in vitro drug release experiments in a simulated gastric fluid (pH = 1.2) and compared the drug-release profiles of AEA-coated SDN–MMT and Viagra®, an approved dosage form of SDN. As a result, about 90% of SDN was released from the AEA-coated SDN–MMT during the first 2 hours while almost 100% of drug was released from Viagra®. However, an in vivo experiment showed that the AEA-coated SDN–MMT exhibited higher drug exposure than Viagra®. For the AEA-coated SDN–MMT, the area under the plasma concentration– time curve from 0 hours to infinity (AUC0-∞) and maximum concentration (Cmax) were 78.8 ± 2.32 μg · hour/mL and 12.4 ± 0.673 μg/mL, respectively, both of which were larger than those obtained with Viagra® (AUC0-∞ = 69.2 ± 3.19 μg · hour/mL; Cmax = 10.5 ± 0.641 μg/mL). Therefore, we concluded that the MMT-based nanohybrid is a promising delivery system for taste masking of SDN with possibly improved drug exposure

Prognostic Significance of Angiogenesis by Chalkley Counting in Node Negative Cancer of the Ampulla of Vater

Angiogenesis is essential for tumor growth and metastasis. Currently, the Chalkley assay with CD34 immunostaining is the proposed standard method for angiogenesis quantification in solid tumor sections. The purpose of this study was to evaluate the expression of CD34 and its prognostic significance using the Chalkley method in node negative carcinoma of the ampulla of Vater. Between January 1997 and December 2006, 56 node negative patients who had curative resection for carcinoma of the ampulla of Vater were retrospectively reviewed. The Chalkley count was expressed as the mean value of the three counts for each tumor and further divided into two groups according to the mean value of the Chalkley count: low < 4 or high ≥ 4. The mean Chalkley count value was 4.0 (± 3.1). In the low Chalkley group, the 1- and 3-yr recurrence rates were 18.3%, 47.6% respectively; in the high Chalkley group, the 1- and 3-yr recurrence rates were 26.5% and 60.6% respectively. Only high Chalkley count had statistical significance as a factor in recurrence of node negative ampulla of Vater carcinoma. Assessment of angiogenesis may have an important role in the prognostic evaluation of node negative cancer of the ampulla of Vater

Whole-genome sequencing and analysis of Streptomyces strains producing multiple antinematode drugs

Background : Nematodes are parasitic animals that cause over 100 billion US dollars loss in agricultural business. The whole-genomes of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced. Both strains produce spectinabilin, an antinematode drug. Its secondary metabolism was examined to aid the development of an efficient nematicidal drug-producing host strain. Results : The whole-genome sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were analyzed using PacBio and Illumina sequencing platforms, and assembled using hybrid methodology. The total contig lengths for KCTC9218T and AN091965 were 9.97 Mb and 9.84 Mb, respectively. A total of 8,374 and 8,054 protein-coding genes, as well as 39 and 45 secondary metabolite biosynthetic gene clusters were identified in KCTC9218T and AN091965, respectively. 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Pine wilt disease caused by nematode was successfully prevented by lower concentration of spectinabilin injection than that of abamectin recommended by its manufacturer. Production of multiple antinematode drugs, including spectinabilin, streptorubin B, and undecylprodigiosin was observed in both strains using high-resolution liquid chromatography mass spectrometry (LC–MS) analysis. Conclusions : Whole-genome sequencing of spectinabilin-producing strains, coupled with bioinformatics and mass spectrometry analyses, revealed the production of multiple nematicidal drugs in the KCTC9218T and AN091965 strains. Especially, Streptomyces sp. AN091965 showed high production level of spectinabilin, and this study provides crucial information for the development of potential nematicidal drug producers.This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through“Crop Viruses and Pests Response Industry Technology Development” Program (No.321110–4) funded by Ministry of Agriculture, Food and Rural Afairs (MAFRA), National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.2022R1A2C3004621) and the Ministry of Education (2022R1I1A1A01068507), and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2020M3H1A1073304)