Macrophage In Vitro and In Vivo Tracking via Anchored Microcapsules.

A new promising trend in personalized medicine is the use of autologous cells (macrophages or stem cells) for cell-based therapy and also as a "Trojan horse" for targeted delivery of a drug carrier. The natural ability of macrophages for chemotaxis allows them to deliver cargo to the damaged area, significantly reducing side effects on healthy organ tissues. Therefore, it is important to develop tools to track their behavior in the organism. While labeled containers can serve as anchored tags for imaging macrophages in vivo, they can affect the properties and functions of macrophages. This work demonstrates that 3 µm sized capsules based on biocompatible polyelectrolytes and fluorescently labeled with both Cy7 and RITC dyes do not affect cell functionalization in vitro, such as viability, proliferation, and movement of transformed monocyte/macrophage-like cells (RAW 264.7) and primary bone marrow derived macrophages (BMDM) at maximal loading of five capsules per cell. In addition, capsules allowed fluorescent detection of ex vivo loaded cells 24 h after the tail vein injection in vivo and visualization of microcapsule-laden macrophages ex vivo using confocal microscopy. We have delivered about 62.5% of injected BMDM containing 12.5 million capsules with 3.75 µg of high-molecular-weight cargo (0.3 pg/capsule) to the liver. Our results demonstrate that 3 µm polyelectrolyte fluorescently labeled microcapsules can be used for safe macrophage loading, allowing cell tracking and drug delivery, which will facilitate development of macrophage-based cell therapy protocols.

Multifunctional nanostructured drug delivery carriers for cancer therapy: Multimodal imaging and ultrasound-induced drug release.

Development of multimodal systems for therapy and diagnosis of neoplastic diseases is an unmet need in oncology. The possibility of simultaneous diagnostics, monitoring, and therapy of various diseases allows expanding the applicability of modern systems for drug delivery. We have developed hybrid particles based on biocompatible polymers containing magnetic nanoparticles (MNPs), photoacoustic (MNPs), fluorescent (Cy5 or Cy7 dyes), and therapeutic components (doxorubicin). To achieve high loading efficiency of MNP and Dox to nanostructured carriers, we utilized a novel freezing-induced loading technique. To reduce the systemic toxicity of antitumor drugs and increase their therapeutic efficacy, we can use targeted delivery followed by the remote control of drug release using high intensity-focused ultrasound (HIFU). Loading of MNPs allowed performing magnetic targeting of the carriers and enhanced optoacoustic signal after controlled destruction of the shell and release of therapeutics as well as MRI imaging. The raster scanning optoacoustic mesoscopy (PA, RSOM), MRI, and fluorescent tomography (FT) confirmed the ultrasound-induced release of doxorubicin from capsules: in vitro (in tubes and pieces of meat) and in vivo (after delivery to the liver). Disruption of capsules results in a significant increase of doxorubicin and Cy7 fluorescence initially quenched by magnetite nanoparticles that can be used for real-time monitoring of drug release in vivo. In addition, we explicitly studied cytotoxicity, intracellular localization, and biodistribution of these particles. Elaborated drug delivery carriers have a good perspective for simultaneous imaging and focal therapy of different cancer types, including liver cancer.

Translation elongation factor 2 depletion by siRNA in mouse liver leads to mTOR-independent translational upregulation of ribosomal protein genes.

Due to breakthroughs in RNAi and genome editing methods in the past decade, it is now easier than ever to study fine details of protein synthesis in animal models. However, most of our understanding of translation comes from unicellular organisms and cultured mammalian cells. In this study, we demonstrate the feasibility of perturbing protein synthesis in a mouse liver by targeting translation elongation factor 2 (eEF2) with RNAi. We were able to achieve over 90% knockdown efficacy and maintain it for 2 weeks effectively slowing down the rate of translation elongation. As the total protein yield declined, both proteomics and ribosome profiling assays showed robust translational upregulation of ribosomal proteins relative to other proteins. Although all these genes bear the TOP regulatory motif, the branch of the mTOR pathway responsible for translation regulation was not activated. Paradoxically, coordinated translational upregulation of ribosomal proteins only occurred in the liver but not in murine cell culture. Thus, the upregulation of ribosomal transcripts likely occurred via passive mTOR-independent mechanisms. Impaired elongation sequesters ribosomes on mRNA and creates a shortage of free ribosomes. This leads to preferential translation of transcripts with high initiation rates such as ribosomal proteins. Furthermore, severe eEF2 shortage reduces the negative impact of positively charged amino acids frequent in ribosomal proteins on ribosome progression.

Oligoglutamylation of E. coli ribosomal protein S6 is under growth phase control.

Ribosomal protein S6 in Escherichia coli is modified by ATP-dependent glutamate ligase RimK. Up to four glutamate residues are added to the C-terminus of S6 protein. In this work we demonstrated that unlike the majority of ribosome modifications in E. coli, oligoglutamylation of S6 protein is regulated and happens only in the stationary phase of bacterial culture. Only S6 protein incorporated into assembled small ribosomal subunits, but not newly made free S6 protein is a substrate for RimK protein. Overexpression of the rimK gene leads to the modification of S6 protein even in the exponential phase of bacterial culture. Thus, it is unlikely that any stationary phase specific factor is needed for the modification. We propose a model that S6 modification is regulated solely via the rate of ribosome biosynthesis at limiting concentration of RimK enzyme.

Phenotypic characterization and complementation analysis of Bacillus subtilis 6S RNA single and double deletion mutants.

6S RNA, a global regulator of transcription in bacteria, binds to housekeeping RNA polymerase (RNAP) holoenzymes to competitively inhibit transcription from DNA promoters. Bacillus subtilis encodes two 6S RNA homologs whose differential functions are as yet unclear. We constructed derivative strains of B. subtilis PY79 lacking 6S-1 RNA (ΔbsrA), 6S-2 RNA (ΔbsrB) or both (ΔbsrAB) to study the physiological role of the two 6S RNAs. We observed two growth phenotypes of mutant strains: (i) accelerated decrease of optical density toward extended stationary phase and (ii) faster outgrowth from stationary phase under alkaline stress conditions (pH 9.8). The first phenotype was observed for bacteria lacking bsrA, and even more pronounced for ΔbsrAB bacteria, but not for those lacking bsrB. The magnitude of the second phenotype was relatively weak for ΔbsrB, moderate for ΔbsrA and again strongest for ΔbsrAB bacteria. Whereas ΔbsrAB bacteria complemented with bsrB or bsrA (strains ΔbsrAB + B and ΔbsrAB + A) mimicked the phenotypes of the ΔbsrA and ΔbsrB strains, respectively, complementation with the gene ssrS encoding Escherichia coli 6S RNA failed to cure the "low stationary optical density" phenotype of the double mutant, despite ssrS expression, in line with previous findings. Finally, proteomics (two-dimensional differential gel electrophoresis, 2D-DIGE) of B. subtilis 6S RNA deletion strains unveiled a set of proteins that were expressed at higher levels particularly during exponential growth and preferentially in mutant strains lacking 6S-2 RNA. Several of these proteins are involved in metabolism and stress responses.

Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C.

Microcin C (McC) is a peptide-nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects-ribosome-induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori.

How much can we learn about the function of bacterial rRNA modification by mining large-scale experimental datasets?

Modification of ribosomal RNA is ubiquitous among living organisms. Its functional role is well established for only a limited number of modified nucleotides. There are examples of rRNA modification involvement in the gene expression regulation in the cell. There is a need for large data set analysis in the search for potential functional partners for rRNA modification. In this study, we extracted phylogenetic profile, genome neighbourhood, co-expression and phenotype profile and co-purification data regarding Escherichia coli rRNA modification enzymes from public databases. Results were visualized as graphs using Cytoscape and analysed. Majority linked genes/proteins belong to translation apparatus. Among co-purification partners of rRNA modification enzymes are several candidates for experimental validation. Phylogenetic profiling revealed links of pseudouridine synthetases with RF2, RsmH with translation factors IF2, RF1 and LepA and RlmM with RdgC. Genome neighbourhood connections revealed several putative functionally linked genes, e.g. rlmH with genes coding for cell wall biosynthetic proteins and others. Comparative analysis of expression profiles (Gene Expression Omnibus) revealed two main associations, a group of genes expressed during fast growth and association of rrmJ with heat shock genes. This study might be used as a roadmap for further experimental verification of predicted functional interactions.