Institutt for farmasi

De novo sequencing of disulphide rich peptides using tandem mass spectrometry

Wed, 13 May 2015 00:00:00 GMT

Chala, Regasa
Peptide based drugs have been shown to have better target selectivity and thereby better safety profile than many small molecule drug candidates. This led many researchers and drug companies to show an increasing interest toward bioactive peptides. Developing an effective analytical method for peptide /protein structural identification is a cornerstone to meet this expanding area of research in this field. De novo sequencing is a method of peptide structural identification without using peptide databases. Compared to the other de novo sequencing methods, mass spectrometric peptide sequence analysis is superior in terms of sensitivity and speed. Peptide structural information is contained in the spectrum produced by tandem mass spectrometers MS/MS. The quality of the data contained in MS/MS spectra depends on the amino acid content of the peptide, the instrumentation and methods used. One commonly used peptide fragmentation method is collision induced dissociation (CID) where the peptide collides with an inert gas. Depending on the amount of energy applied during fragmentation, peptides yield a wide array of product ions. Peptides of known amino acid sequence were used to determine the relationship between precursor ion mass-to- charge-ratio (m/z) and collision energy needed for optimal peptide fragmentation. The energy seems to increase with increasing m/z of the precursor ion. A comparison of CID fragmentation methods in a quadrupole-time-of-flight (QTof) instrument revealed the merit of using a collision energy ramp compared to single collision energy. Evaluation of de novo sequencing accuracy of PEAKS software showed that the program works well for smaller peptides but the accuracy deteriorates with increasing peptide length. The amino acid sequence of an unknown peptide was determined using the PEAKS. The software has identified 84 % the amino acid sequences correctly. The rest 16 % of the residues were confirmed by Edman degradation. The peptide contained three disulphide bridges and identifying cysteine interconnectivity of the peptide is a gap to be addressed in the future. Key words: Collision energy, ramp, PEAKS, de novo sequencing.

Costs and benefits of natural transformation in Acinetobacter baylyi

Wed, 15 Feb 2017 00:00:00 GMT

Hülter, Nils Fredrik; Sørum, Vidar; Borch-Pedersen, Kristina; Liljegren, Mikkel Meyn; Utnes, Ane; Primicerio, Raul; Harms, Klaus; Johnsen, Pål Jarle
Background
Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells.
Results
We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation. Conclusions:
Our data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation.
Source at http://dx.doi.org/10.1186/s12866-017-0953-2

Interplay of choline metabolites and genes in patient-derived breast cancer xenografts

Tue, 21 Jan 2014 00:00:00 GMT

Grinde, Maria Tunset; Skrbo, Nirma; Moestue, Siver Andreas; Rødland, Einar Andreas; Borgan, Eldrid; Kristian, Alexandr; Sitter, Beathe; Bathen, Tone; Børresen-Dale, Anne-Lise; Mælandsmo, Gunhild; Sørlie, Therese; Marangoni, Elisabetta; Gribbestad, Ingrid S
Introduction: Dysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism.
Methods: Tumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson’s correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups.
Results: Most of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples.
Conclusion: The higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo.
Source at http://dx.doi.org/10.1186/bcr3597 .

Successful co-encapsulation of benzoyl peroxide and chloramphenicol in liposomes by a novel manufacturing method - dual asymmetric centrifugation

Thu, 16 Nov 2017 00:00:00 GMT

Ingebrigtsen, Sveinung Gaarden; Skalko-Basnet, Natasa; Cavalcanti Jacobsen, Cristiane de Albuquerque; Holsæter, Ann Mari
Encapsulation of more than one active pharmaceutical ingredient into nanocarriers such as liposomes is an attractive approach to achieve a synergic drug effect and less complicated dosing schedules in multi-drug treatment regimes. Liposomal drug delivery in acne treatment may improve drug efficiency by targeted delivery to pilosebaceous units, reduce adverse effects and improve patient compliance. We therefore aimed to co-encapsulate benzoyl peroxide (BPO) and chloramphenicol (CAM) into liposomes using the novel liposome processing method – dual asymmetric centrifugation (DAC). Liposomes were formed from soybean lecithin, propylene glycol and distilled water (2:1:2 w/v/v ratio), forming a viscous liposome dispersion. Liposomes containing both drugs (BPO-CAM-Lip), single-drug (BPO-Lip and CAM-Lip), and empty liposomes were prepared. Drug entrapment of BPO and CAM was determined by a newly developed HPLC method for simultaneous detection and quantification of both drugs. Encapsulation of around 50% for BPO and 60% for CAM respectively was obtained in both single- drug encapsulated formulations (BPO-Lip and CAM-Lip) and co-encapsulated formulations (BPO- CAM-Lip). Liposome sizes were comparable for all liposome formulations, ranging from 130 to 150 nm mean diameter, with a polydispersity index < 0.2 for all formulations. CAM exhibited a sustained release from all liposomal formulations, whereas BPO appeared retained within the liposomes. BPO retention could be attributed to its poor solubility. However, HaCaT cell toxicity was found dependent on BPO released from the liposomes. In the higher concentration range (4% v/v), liposomal formulations were less cytotoxic than the corresponding drug solutions used as reference. We have demonstrated that DAC is a fast, easy, suitable method for encapsulation of more than one drug within the same liposomes.
Accepted manuscript version. Published version available at http://dx.doi.org/10.1016/j.ejps.2016.11.017

The Potential of Cyclodextrins as Novel Active Pharmaceutical Ingredients: A Short Overview

Sun, 25 Dec 2016 00:00:00 GMT

Di Cagno, Massimiliano Pio
Cyclodextrins (CDs) are cyclic oligosaccharides of natural origin that were discovered more than 100 years ago. The peculiar cone-like conformation of the sugar ring, expressing a lipophilic cavity and a hydrophilic external surface, allows these substances to spontaneously complex poorly soluble compounds in an aqueous environment. For more than 50 years, these substances have found applicability in the pharmaceutical and food industries as solubilizing agents for poorly soluble chemical entities. Nowadays, several research groups all over the world are investigating their potential as active pharmaceutical ingredients (APIs) for the treatment of several illnesses (e.g., hypercholesterolemia, cancer, Niemann-Pick Type C disease). The aim of this review is to briefly retrace cyclodextrins’ legacy as complexing agents and describe the current and future prospects of this class of chemical entities in pharmaceutics as new APIs.
Published version. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-SA 4.0). Source at http://dx.doi.org/10.3390/molecules22010001.

No effect of natural transformation on the evolution of resistance to bacteriophages in the Acinetobacter baylyi model system.

Fri, 01 Jan 2016 00:00:00 GMT

Mcleman, Amy; Sierocinski, Pawel; Hesse, E; Buckling, A; Perron, G; Hülter, Nils Fredrik; Johnsen, Pål Jarle; Vos, M
The adaptive benefits of natural transformation, the active uptake of free DNA molecules from the environment followed by incorporation of this DNA into the genome, may be the improved response to selection resulting from increased genetic variation. Drawing analogies with sexual reproduction, transformation may be particularly beneficial when selection rapidly fluctuates during coevolution with virulent parasites (‘the Red Queen Hypothesis’). Here we test this hypothesis by experimentally evolving the naturally transformable and recombinogenic species Acinetobacter baylyi with a cocktail of lytic phages. No increased levels of resistance to phage were found in the wild type compared to a recombination deficient ΔdprA strain after five days of evolution. When exposed to A. baylyi DNA and phage, naturally transformable cells show greater levels of phage resistance. However, increased resistance arose regardless of whether they were exposed to DNA from phage-sensitive or –resistant A. baylyi, suggesting resistance was not the result of transformation, but was related to other benefits of competence. Subsequent evolution in the absence of phages did not show that recombination could alleviate the cost of resistance. Within this study system we found no support for transformation-mediated recombination being an advantage to bacteria exposed to parasitic phages.
Published version. Source at http://doi.org/10.1038/srep37144. License CC BY 4.0.

The Role of Adherence and Retreatment in De Novo Emergence of MDR-TB

Fri, 01 Jan 2016 00:00:00 GMT

Cadosch, Dominique; Abel zur Wiesch, Pia; Kouyos, Roger; Bonhoeffer, Sebastian
Treatment failure after therapy of pulmonary tuberculosis (TB) infections is an important challenge, especially when it coincides with de novo emergence of multi-drug-resistant TB (MDR-TB). We seek to explore possible causes why MDR-TB has been found to occur much more often in patients with a history of previous treatment. We develop a mathematical model of the replication of Mycobacterium tuberculosis within a patient reflecting the compartments of macrophages, granulomas, and open cavities as well as parameterizing the effects of drugs on the pathogen dynamics in these compartments. We use this model to study the influence of patient adherence to therapy and of common retreatment regimens on treatment outcome. As expected, the simulations show that treatment success increases with increasing adherence. However, treatment occasionally fails even under perfect adherence due to interpatient variability in pharmacological parameters. The risk of generating MDR de novo is highest between 40% and 80% adherence. Importantly, our simulations highlight the double-edged effect of retreatment: On the one hand, the recommended retreatment regimen increases the overall success rate compared to re-treating with the initial regimen. On the other hand, it increases the probability to accumulate more resistant genotypes. We conclude that treatment adherence is a key factor for a positive outcome, and that screening for resistant strains is advisable after treatment failure or relapse.
Published version. Source at http://doi.org/10.1371/journal.pcbi.1004749. License CC BY 4.0.

The Presumed Polyomavirus Viroporin VP4 of Simian Virus 40 or Human BK Polyomavirus Is Not Required for Viral Progeny Release

Wed, 14 Sep 2016 00:00:00 GMT

Henriksen, Stian; Hansen, Terkel; Bruun, Jack-Ansgar; Rinaldo, Christine Hanssen
The minor capsid protein of human BK polyomavirus (BKPyV), VP2, and its N-terminally truncated form, VP3, are both important for viral entry. The closely related simian virus 40 (SV40) reportedly produces an additional truncated form of VP2/3, denoted VP4, apparently functioning as a viroporin promoting progeny release. The VP4 open reading frame is conserved in some polyomaviruses, including BKPyV. In this study, we investigated the role of VP4 in BKPyV replication. By transfecting viral genomes into primary human renal proximal tubule epithelial cells, we demonstrated that unaltered BKPyV and mutants with start codon substitutions in VP4 (VP2M229I and VP2M229A) abolishing putative VP4 production were released at the same level to supernatants. However, during infection studies, VP2M229I and VP2M229A exhibited 90% and 65% reduced infectivity, respectively, indicating that isoleucine substitution inadvertently disrupted VP2/3 function to the detriment of viral entry, while inhibition of VP4 production during late infection was well tolerated. Unexpectedly, and similarly to BKPyV, wild-type SV40 and the corresponding VP4 start codon mutants (VP2M228I and VP2M228A) transfected into monkey kidney cell lines were also released at equal levels. Upon infection, only the VP2M228I mutant exhibited reduced infectivity, a 43% reduction, which also subsequently led to delayed host cell lysis. Mass spectrometry analysis of nuclear extracts from SV40-infected cells failed to identify VP4. Our results suggest that neither BKPyV nor SV40 require VP4 for progeny release. Moreover, our results reveal an important role in viral entry for the amino acid in VP2/VP3 unavoidably changed by VP4 start codon mutagenesis.
Source:doi:10.1128/JVI.01326-16.

Reconstruction of a nonlinear heat transfer law from uncomplete boundary data by means of infrared thermography

Fri, 07 Oct 2016 00:00:00 GMT

Clarelli, Fabrizio; Inglese, Gabriele
Heat exchange between a conducting plate and the environment is described here by means of an unknown nonlinear function F of the temperature u. In this paper we construct a method for recovering F by means of polynomial expansion, perturbation theory and the toolbox of thermal inverse problems. We test our method on two examples: In the first one, we heat the plate (initially at 20 °C) from one side, read the temperature on the same side and identify the heat exchange law on the opposite side (active thermography); in the second example we measure the temperature of one side of the plate (initially at 1500 °C) and study the heat exchange while cooling (passive thermography)
Manuscript. Published version available in Inverse Problems, 2016, vol. 32, no.11. doi: 10.1088/0266-5611/32/11/115017

Fibroblast-induced switching to the mesenchymal-like phenotype and PI3K/mTOR signaling protects melanoma cells from BRAF inhibitors

Fri, 01 Jan 2016 00:00:00 GMT

Vasiliauskaite, Kotryna; Fleten, Karianne Giller; Barkovskaya, Anna; Nygaard, Vigdis; Haugen, Mads Haugland; Engesæter, Birgit Øvstebø; Mælandsmo, Gunhild; Prasmickaite, Lina
The knowledge on how tumor-associated stroma influences efficacy of anti-cancer therapy just started to emerge. Here we show that lung fibroblasts reduce melanoma sensitivity to the BRAF inhibitor (BRAFi) vemurafenib only if the two cell types are in close proximity. In the presence of fibroblasts, the adjacent melanoma cells acquire de-differentiated mesenchymal-like phenotype. Upon treatment with BRAFi, such melanoma cells maintain high levels of phospho ribosomal protein S6 (pS6), i.e. active mTOR signaling, which is suppressed in the BRAFi sensitive cells without stromal contacts. Inhibitors of PI3K/mTOR in combination with BRAFi eradicate pS6high cell subpopulations and potentiate anti-cancer effects in melanoma protected by the fibroblasts. mTOR and BRAF co-inhibition also delayed the development of early-stage lung metastases in vivo. In conclusion, we demonstrate that upon influence from fibroblasts, melanoma cells undergo a phenotype switch to the mesenchymal state, which can support PI3K/mTOR signaling. The lost sensitivity to BRAFi in such cells can be overcome by co-targeting PI3K/mTOR. This knowledge could be explored for designing BRAFi combination therapies aiming to eliminate both stroma-protected and non-protected counterparts of metastases.
Published version. Source at http://doi.org/10.18632/oncotarget.7671. License CC BY 3.0.