Impact of cell cycle dynamics on pathology recognition: Raman imaging study

Confocal Raman imaging combined with fluorescence‐activated cell sorting was used for in vitro studies of cell cultures to look at biochemical differences between the cells in different cell phases. To answer the question what is the impact of the cell cycle phase on discrimination of pathological cells, the combination of several factors was checked: a confluency of cell culture, the cell cycle dynamics and development of pathology. Confluency of 70% and 100% results in significant phenotypic cell changes that can be also diverse for different batches. In 100% confluency cultures, cells from various phases become phenotypically very similar and their recognition based on Raman spectra is not possible. For lower confluency, spectroscopic differences can be found between cell cycle phases (G₀/G₁, S and G₂/M) for control cells and cells incubated with tumor necrosis factor alpha (TNF‐α), but when the mycotoxin cytochalasin B is used the Raman signatures of cell phases are not separable. Generally, this work shows that heterogeneity between control and inflamed cells can be bigger than heterogeneity between cell cycle phases, but it is related to several factors, and not always can be treated as a rule.      

Urinary proteomic biomarkers in oncology: ready for implementation?

Introduction: Biomarkers are expected to improve the management of cancer patients by enabling early detection and prediction of therapeutic response. Proteins reflect a molecular phenotype, have high potential as biomarkers, and also are key targets for intervention. Given the ease of collection and proximity to certain tumors, the urinary proteome is a rich source of biomarkers and several proteins have been already implemented.

Areas covered: We examined the literature on urine proteins and proteome analysis in oncology from reports published during the last 5 years to generate an overview on the status of urine protein and peptide biomarkers, with emphasis on their actual clinical value.

Expert commentary: A few studies report on biomarkers that are ready to be implemented in patient management, among others in bladder cancer and cholangiocarcinoma. These reports are based on multi-marker approaches. A high number of biomarkers, though, has been described in studies with low statistical power. In fact, several of them have been consistently reported across different studies. The latter should be the focus of attention and be tested in properly designed confirmatory and ultimately, prospective investigations. It is expected that multi-marker classifiers for a specific context-of-use, will be the preferred path toward clinical implementation.     

Comparison of eight commercially available kits for DNA extraction from formalin-fixed paraffin-embedded tissues

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA.     

Biotransformation of cinnamyl alcohol to rosavins by non-transformed wild type and hairy root cultures of Rhodiola kirilowii

Non-transformed wild type (NTWT) and hairy root cultures of Rhodiola kirilowii were grown in medium supplemented with 2.5 mM cinnamyl alcohol as a precursor and/or sucrose (1 %) on the day of inoculation or on the 14th day of culture. Rosarin, rosavin, and rosin were produced by NTWT root culture but only rosarin and rosavin by hairy roots. Approximately 80 and 95 % of the glycosides were released into the medium for NTWT and hairy root cultures, respectively. The highest rosavin yield, 505 ± 106 mg/l, was in hairy root culture when cinnamyl alcohol was applied on the day of inoculation with the addition of sucrose on the 14th day of culture. For rosin production, supplementation with cinnamyl alcohol alone on day 14 was more favourable with the highest amount 74 ± 10 mg/l in NTWT root culture. Only traces of rosarin were detected.