Stability study of sodium colistimethate-loaded lipid nanoparticles.

In the last decades, the encapsulation of antibiotics into nanoparticulate carriers has gained increasing attention for the treatment of infectious diseases. Sodium colistimethate-loaded solid lipid nanoparticles (Colist-SLNs) and nanostructured lipid carriers (Colist-NLCs) were designed aiming to treat the pulmonary infection associated to cystic fibrosis patients. The nanoparticles were freeze-dried using trehalose as cryoprotectant. The stability of both nanoparticles was analysed over one year according to the International Conference of Harmonisation (ICH) guidelines by determining the minimum inhibitory concentration (MIC) against clinically isolated Pseudomonas aeruginosa strains and by studying their physico-chemical characteristics. The results showed that Colist-SLNs lost their antimicrobial activity at the third month; on the contrary, the antibacterial activity of Colist-NLCs was maintained throughout the study within an adequate range (MIC ≤16 µg/mL). In addition, Colist-NLCs exhibited suitable physico-chemical properties at 5 °C and 25 °C/60% relative humidity over one year. Altogether, Colist-NLCs proved to have better stability than Colist-SLNs.

Killing effect of nanoencapsulated colistin sulfate on Pseudomonas aeruginosa from cystic fibrosis patients.

Pseudomonas aeruginosa frequently infects the respiratory tract of cystic fibrosis (CF) patients. Multidrug-resistant phenotypes and high capacity to form stable biofilms are common. Recent studies have described the emergence of colistin-resistant isolates in CF patients treated with long-term inhaled colistin. The use of nanoparticles containing antimicrobials can contribute to overcome drug resistance mechanisms. The aim of this study was to explore antimicrobial activity of nanoencapsulated colistin (SLN-NLC) versus free colistin against P. aeruginosa clinical isolates from CF patients and to investigate their efficacy in biofilm eradication. Susceptibility of planktonic bacteria to antimicrobials was examined by using the broth microdilution method and growth curve assay. Minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) were determined to assess antimicrobial susceptibility of sessile bacteria. We used atomic force microscopy (AFM) to visualize treated and untreated biofilms and to determine surface roughness and other relevant parameters. Colistin nanoparticles had the same antimicrobial activity as free drug against planktonic bacteria. However, nanoencapsulated colistin was much more efficient in the eradication of biofilms than free colistin. Thus, these formulations have to be considered as a good alternative therapeutic option to treat P. aeruginosa infections.

Generation of mouse and human induced pluripotent stem cells (iPSC) from primary somatic cells.

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663-676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949-953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543-549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348-362, 2009), removal of the reprogramming vectors by 'piggyBac' transposition Woltjen et al. (Nature 458(7239):766-770, 2009); Kaji et al. (Nature 458(7239):771-775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964-977, 2009), episomal vectors Yu et al. (Science 324(5928):797-801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381-384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376-388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789-797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human somatic cells and their reprogramming into iPSC. All-encompassing instructions, not previously published in a single document, are provided for mouse and human iPSC colony isolation and derivation. Although mouse and human iPSC share similarities in the cellular reprogramming process and culture, both cell types need to be handled differently.

BMP-binding modules in chordin: a model for signalling regulation in the extracellular space.

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling.

Deregulated c-fos modulates IgG2b production of B cells mediated by lipopolysaccharide.

We have examined effects of the deregulated c-fos protein on IgG2b production of B cells cultured with lipopolysaccharide (LPS) using splenic B cells from a transgenic line carrying the mouse c-fos gene under the control of the interferon alpha/beta (IFN) inducible Mx promoter (Mx-c-fosD). High c-fos expression was induced in the Mx-c-fosD B cells during the first two days of culture. DNA synthesis and IgG2b production were augmented in the culture. When IFN was added together with LPS, the high c-fos expression was prolonged until day 3 of culture. IgG2b production was remarkably suppressed. However, the production was not suppressed by upregulation of c-fos via exogenous IFN on day 4 of culture. These results suggest a regulatory effect of the c-fos protein on the differentiation of B cells to IgG2b producing cells at a distinct period.

Deregulated c-fos augments cell proliferation of B cells mediated by lipopolysaccharide.

We have examined effects of the deregulated c-fos protein on the lipopolysaccharide (LPS)-mediated B cell responses using splenic B cells from transgenic lines carrying the mouse c-fos gene under the control of the H-2K (H2-c-fos) and the inducible Mx promoter (Mx-c-fosD). High c-fos expression was induced in Mx-c-fosD B cells by LPS stimulation. DNA synthesis of the B cells from both lines was augmented depending on the amount of exogenous c-fos. This augmentation resulted in the increase of IgM and IgG2b productions in the culture. These results suggest a functional role of c-fos protein in cell cycle progression of the activated B cells.

Inducible expression of the proto-oncogene c-fos in transgenic mice.

We have generated transgenic mouse lines expressing the proto-oncogene c-fos under the control of the interferon inducible Mx promotor. We found low expression in skeletal muscle, brain and salivary glands and a very high expression in spleen, liver, thymus, heart and kidney. Despite this transgenic c-fos expression we have not yet detected any phenotypic changes in these mouse lines.