f Pseudomonas aeruginosa Liverpool epidemic strain (LES) infections in cystic fibrosis (CF) patients are associated with transmissibility and increased patient morbidity. This study was designed to assess the in vitro activities of cathelicidin LL-37 peptide (LL-37) and select cationic lipids against Pseudomonas aeruginosa LESB58 in CF sputum and in a setting mimicking the CF airway. We found that LL-37 naturally present in airway surface fluid and some nonpeptide cationic lipid molecules such as CSA-13, CSA-90, CSA-131, and D2S have significant, but broadly differing, bactericidal activities against P. aeruginosa LESB58. We observed strong inhibition of LL-37 bactericidal activity in the presence of purified bacteriophage Pf1, which is highly expressed by P. aeruginosa LES, but the activities of the cationic lipids CSA-13 and CSA-131 were not affected by this polyanionic virus. Additionally, CSA-13 and CSA-131 effectively prevent LESB58 biofilm formation, which is stimulated by Pf1 bacteriophage, DNA, or F-actin. CSA-13 and CSA-131 display strong antibacterial activities against different clinical strains of P. aeruginosa, and their activities against P. aeruginosa LESB58 and Xen5 strains were maintained in CF sputum. These data indicate that synthetic cationic lipids (mimics of natural antimicrobial peptides) are suitable for developing an effective treatment against CF lung P. aeruginosa infections, including those caused by LES strains. Pseudomonas aeruginosa chronic lung infections are the major cause of death in cystic fibrosis (CF) patients. The identification of the Liverpool epidemic strain (LES) in numerous children's CF centers challenged the previous belief that CF patients acquire only unique environmental strains of P. aeruginosa (1). LES infections were found to be associated with transmissibility (2), dominance over other P. aeruginosa populations in CF airways, increased patient morbidity (3), and frequent infections in parents of children with CF (4, 5). LES isolates display widely variable pathogenic characteristics (6). LES sequencing revealed that multiple inducible prophages with diverse infection properties have been conserved within the bacterial DNA (4, 7). When studied in a rat model of chronic lung infection, P. aeruginosa strains such as PAO1, PA14, and LESB58 demonstrated similar levels of in vivo growth but differences in virulence. These differences were further confirmed with biofilm and motility assays, where LESB58 produced more biofilm but had less capacity for motility than PAO1 and PA14 (8). Taking into consideration that LES isolates exhibit enhanced antimicrobial resistance, which is not fully understood but linked to specific mutations in efflux pump genes (9) and premature activation of quorum-sensing exoproducts (10), we hypothesized that cationic antibacterial peptides (CAPs) and their synthetic mimics, which use a nonspecific physicochemical mode of bacterial killing (11, 12), might represent promising new methods for eradicating this bacterium from lung infections. In...

MicroRNAs (miRNAs) can control stem cell differentiation by targeting mRNAs. Using 96-well plate electroporation, we screened 466 human miRNA mimics by 4-color flow cytometry to explore differentiation of common myeloid progenitors (CMP) derived from human embryonic stem cells (hESCs). The transfected cells were then cultured in a cytokine cocktail that supported multiple hematopoietic lineages. At 4-5 days post-transfection, flow cytometry of erythroid (CD235+CD41−), megakaryocyte (CD41+CD42+) and myeloid (CD18+CD235−) lineages revealed miR-105 as a novel enhancer of megakaryocyte production during in vitro primitive hematopoiesis. In hESC-derived CMPs, miR-105 caused a 6-fold enhancement in megakaryocyte production. MiR-513a, miR-571 and miR-195 were found to be less potent megakaryocyte enhancers. We confirmed the relevance of miR-105 in adult megakaryopoiesis by demonstrating increased megakaryocyte yield and megakaryocyte colony forming potential in human adult CD34+ cells derived from peripheral blood. In addition, adult CD34+ cells express endogenous miR-105 during megakaryocyte differentiation. SiRNA knockdown of the hematopoietic transcription factor c-Myb caused a similar enhancement of megakaryocyte production as miR-105. Finally, a luciferase/c-Myb-3’UTR construct and western blot analysis demonstrated that the hematopoietic transcription factor c-Myb mRNA was a target of miR-105. We report a novel hESC-based miR screening platform and demonstrate that miR-105 is an enhancer of megakaryopoiesis in both primitive and definitive hematopoiesis.

CD8 + T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8 + T cell responses, therapeutic approaches to generate Ag-specific CD8 + T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8 + T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ~1000-fold more potent at eliciting CD8 + T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8 + T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8 + T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8 + T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8 + T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.

The partitioning of pristinamycins was studied in dextran and polyethylene glycol (PEG) aqueous two-phases systems. Pristinamycins partitioned preferentially into the PEG-rich top phase. The partition coefficient was independent of molar mass of PEG and dextran and of antibiotic concentration, but, increased exponentially with the tieline length of the system. Partition of pristinamycins was greatly improved when fatty acids esters of PEG were mixed with PEG. In such mixtures, the partition of coefficient increased up to a value of 24, dependent on the carbon chain length of fatty acids and the modified PEG concentrations. Moreover, in such system, the two groups of pristinamycins, I and II, were extracted in accordance with their hydrophobicity. Recovery of pristinanamycins produced by Streptomyces pritinaespiralis in a fermentation broth was achieved with a dextran/PEG system. Cells were confined into the bottom phase and pristinamycins partitioned in the top phase. However, due to binding of the pristinamycins to the cells, the partition coefficient was slightly lower than of pure antibiotics solutions. (c) 1994 John Wiley & Sons, Inc.

Aims:  Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results:  Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions:  Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study:  Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.