Heterologous expression of tylosin polyketide synthase and production of a hybrid bioactive macrolide in Streptomyces venezuelae

Tylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts. To improve the production level of tylactone, several precursors for ethylmalonyl-CoA were fed to the growing medium, leading to a 2.8-fold improvement (1.4 mg/ml); however, switching the pikAI promoter to an actI promoter had no observable effect. In addition, a small amount of desosamine-glycosylated tylactone was detected from the extract of the mutant strain, revealing that the native glycosyltransferase DesVII displayed relaxed substrate specificity in accepting the 16-membered ring macrolactone to produce the glycosylated tylactone. These results demonstrate a successful attempt for a heterologous expression of Tyl PKS in S. venezuelae and introduce S. venezuelae as a rapid heterologous expression system for the production of secondary metabolites.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45861/1/253_2006_Article_318.pd

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling

Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5' RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5' UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5' UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5' UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization

Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features

Background: The increasing number of infections caused by strains of Klebsiella pneumoniae that are resistant to multiple antibiotics has developed into a major medical problem worldwide. The development of next-generation sequencing technologies now permits rapid sequencing of many K. pneumoniae isolates, but sequence information alone does not provide important structural and operational information for its genome. Results: Here we take a systems biology approach to annotate the K. pneumoniae MGH 78578 genome at the structural and operational levels. Through the acquisition and simultaneous analysis of multiple sample-matched -omics data sets from two growth conditions, we detected 2677, 1227, and 1066 binding sites for RNA polymerase, RpoD, and RpoS, respectively, 3660 RNA polymerase-guided transcript segments, and 3585 transcription start sites throughout the genome. Moreover, analysis of the transcription start site data identified 83 probable leaderless mRNAs, while analysis of unannotated transcripts suggested the presence of 119 putative open reading frames, 15 small RNAs, and 185 antisense transcripts that are not currently annotated. Conclusions: These findings highlight the strengths of systems biology approaches to the refinement of sequence-based annotations, and to provide new insight into fundamental genome-level biology for this important human pathogen

Different organization of upstream regulatory region between <i>E. coli</i> and <i>K. pneumoniae</i>.

<p>(A) Venn diagram showing orthologous genes and species-specific genes between <i>E. coli</i> and <i>K. pneumoniae</i>. (B) 4 different types of promoter regions, and their numbers identified in two species. (C) Schematic drawing of annotated TSSs and sequence comparison of regulatory region upstream of <i>lpd</i>. (D) Length difference between the pairs of comparable 5′ UTR. (E) Comparison of sequence conservation of promoter, 5′ UTR, and ORF regions. (F) Sequence conservation of genomic regions surrounding translation start sites.</p

TSS annotation and structure of promoter region and 5′ UTR.

<p>(A) Number of TSSs assigned per annotated genes. (B) Distribution of 5′ UTR lengths for <i>E. coli</i> and <i>K. pneumoniae</i>, and the Shine-Dalgarno sequence motif. (C) Sequence motif of promoter region containing −10 and −35 boxes. (D) Conservation of RpoD amino acid sequences of 5 species in gammaproteobacteria and 3 other species belonging to proteobacteria. (E) Di-nucleotide preference near the TSS site.</p

Comparison analysis of orthologous sRNAs.

<p>(A) Expression of RNA-binding protein <i>hfq</i> (B) Sequence conservation of regulatory region upstream of <i>hfq</i> ORF, including promoter, TSS and 5′ UTR. (C) Conservation and expression of non-coding regulatory sRNAs, <i>rprA</i>, <i>arcZ</i> and <i>sgrS</i>. (D) Sequence comparison analysis of <i>rprA</i> and <i>arcZ</i> regulating translation of <i>rpoS</i>. (E) Sequence comparison analysis of <i>sgrS</i> regulating translation of <i>ptsG</i> and <i>manX</i>.</p

Experimentally determined TSSs and their association with annotated genes.

<p>(A) Genome-wide TSS mapped onto <i>E. coli</i> and <i>K. pneumoniae</i> genome annotation. (B) Number of <i>E. coli</i> sRNAs detected with 5 TSS datasets generated by different methods. (C) Number of sRNAs detected from <i>E. coli</i> and <i>K. pneumoniae</i> during the exponential growth. (D) Schematic drawing of annotated TSSs assigned to orthologous <i>micF</i> sRNA and coding genes surrounding <i>micF</i> in <i>E. coli</i> and <i>K. pneumoniae</i>. (E) Schematic drawing of annotated TSSs assigned to <i>K. pneumoniae</i> sRNA, <i>rnai</i>, and coding genes near <i>rnai</i>.</p

Leptomeningeal Collaterals and Infarct Progression in Patients With Acute Large‐Vessel Occlusion and Low NIHSS

Background Approximately 10% of patients with acute ischemic stroke with large‐vessel occlusion (LVO) have mild neurological deficits. Although leptomeningeal collaterals (LMCs) are the major determinant of clinical outcomes for patients with acute ischemic stroke with LVO, the contribution of baseline LMC status to subsequent infarct progression in patients with mild stroke with LVO is poorly defined. Methods This observational study included patients with acute anterior circulation LVO and mild stroke symptoms (National Institutes of Health Stroke Scale < 6) from a prospectively collected, multicenter, national stroke registry. The Alberta Stroke Program Early Computed Tomography Score was quantified on the initial and follow‐up images. An infarct progression, defined as any Alberta Stroke Program Early Computed Tomography Score decrease between the initial versus follow‐up scans, was categorized as either 0/1/2+. The LMCs on the baseline images were graded as good, fair, or poor. Results Of the 623 included patients (mean age, 67.6±13.4 years; 380 [61.0%] men; 186 [29.9%] with reperfusion treatment), the baseline LMC was graded as good in 331 (53.1%), fair in 219 (35.2%), and poor in 73 (11.7%). The Alberta Stroke Program Early Computed Tomography Score decrement was noted as 0 in 288 (46%) patients, 1 in 154 (24%), and 2+ in 181 (29%). A poor LMC was associated with an infarct progression (adjusted odds ratio, 2.05 [95% CI, 1.22–3.47]). Conclusions Poor collateral blood flow was associated with infarct progression in patients with acute ischemic stroke with LVO and mild symptoms. In this selective population, early assessment of collateral blood flow status can help in early detection of patients susceptible to infarct progression

Dual antiplatelet Use for extended period taRgeted to AcuTe ischemic stroke with presumed atherosclerotic OrigiN (DURATION) trial : Rationale and design

Rationale: The optimal duration of dual antiplatelet therapy (DAPT) with clopidogrel-aspirin for the large artery atherosclerotic (LAA) stroke subtype has been debated. Aims: To determine whether the 1-year risk of recurrent vascular events could be reduced by a longer duration of DAPT in patients with the LAA stroke subtype. Methods and study design: A total of 4806 participants will be recruited to detect a statistically significant relative risk reduction of 22% with 80% power and a two-sided alpha error of 0.05, including a 10% loss to follow-up. This is a registry-based, multicenter, prospective, randomized, open-label, blinded end point study designed to evaluate the efficacy and safety of a 12-month duration of DAPT compared with a 3-month duration of DAPT in the LAA stroke subtype. Patients will be randomized (1:1) to either DAPT for 12 months or DAPT for 3 months, followed by monotherapy (either aspirin or clopidogrel) for the remaining 9 months. Study outcomes: The primary efficacy outcome of the study is a composite of stroke (ischemic or hemorrhagic), myocardial infarction, and all-cause mortality for 1 year after the index stroke. The secondary efficacy outcomes are (1) stroke, (2) ischemic stroke or transient ischemic attack, (3) hemorrhagic stroke, and (4) all-cause mortality. The primary safety outcome is major bleeding. Discussion: This study will help stroke physicians determine the appropriate duration of dual therapy with clopidogrel-aspirin for patients with the LAA stroke subtype. Trial registration: URL: https://cris.nih.go.kr/cris. CRIS Registration Number: KCT0004407