Optimized dual assay for the transgenes selection and screening in CHO cell line development for recombinant protein production.

OBJECTIVE: To develop a simple robust methodology of screening multiple CHO cell clones secreting recombinant proteins to assess their specific productivity. RESULTS: We developed a dual assay based on immunoassay measurements of a recombinant protein expression combined with staining of viable cells with resazurin. Following this approach, colonies can be simultaneously assessed for cell growth rate and for production of a recombinant protein. Combination of these two assays enables to estimate productivity of a recombinant protein per cell from the very early stages of a cell line development process (CLD) and exclude poor producers from further steps. Comparison of the dual assay with a standard CLD protocol followed by only analysis of protein expression level showed at least 10-20% increase in the amount of clones that can be included into pool of high-producers at early stages. This shortens duration of a typical CLD scheme from 23 to 19 weeks. CONCLUSIONS: Our method: (i) allows to include into workflow clones that demonstrate slow growth during single cell cloning but producing high amounts of a target protein, which otherwise would be lost in standard protocols of cells screening; (ii) can be applied for testing of DNA vectors for transfection and protein production; (iii) can be used for monitoring the heterogeneity of cell population and analysis of stable pools productivity.

Disassembling a cancer puzzle: Cell junctions and plasma membrane as targets for anticancer therapy.

Despite an enhanced permeability and retention effect typical of many solid tumors, drug penetration is not always sufficient. Possible strategies for the drug delivery improvement are a modification of the tumor cell-to-cell junctions and usage of cell membrane permeabilization proteins. In this review we discuss epithelial cell junctions as targets for a combined anticancer therapy and propose new possible sources of such agents. We suggest considering viral and bacterial pathogens disrupting epithelial layers as plentiful sources of new therapeutic agents for increasing tumor permeability for other effector agents. We also observe the application of pore forming proteins and peptides of different origin for cytoplasmic delivery of anti-cancer agents and consider the main obstacles of their use in vivo.

The effect of trypan blue treatment on autofluorescence of fixed cells.

Controlling background fluorescence remains an important challenge in flow cytometry, as autofluorescence can interfere with the detection of chromophores. Furthermore, experimental procedures can also affect cellular fluorescence in certain regions of the emission spectrum. In this work, the effects of fixation, permeabilization, and heating on cellular autofluorescence are analyzed in various spectral regions, along with the influence of trypan blue as a quenching dye for these treatments. The impact of these procedures on the staining of SK-BR-3 cells with a dim green fluorophore, a miniSOG (mini Singlet Oxygen Generator) flavoprotein in the form of the recombinant protein DARPin-miniSOG, is also evaluated. The data presented here indicate that fixation of certain types of cells leads to noticeable increase of the autofluorescence. Our results also suggest that trypan blue should be used as an autofluorescence quencher only with bright green emitters since it interferes with the fluorescent signal in a longer-wavelength region of the spectrum and as a result causes reduction of the signal from dim green fluorescent agents. © 2017 International Society for Advancement of Cytometry.

Nitric Oxide Has a Concentration-Dependent Effect on the Cell Cycle Acting via EIN2 in Arabidopsis thaliana Cultured Cells.

Ethylene is known to influence the cell cycle (CC) via poorly characterized roles whilst nitric oxide (NO) has well-established roles in the animal CC but analogous role(s) have not been reported for plants. As NO and ethylene signaling events often interact we examined their role in CC in cultured cells derived from Arabidopsis thaliana wild-type (Col-0) plants and from ethylene-insensitive mutant ein2-1 plants. Both NO and ethylene were produced mainly during the first 5 days of the sub-cultivation period corresponding to the period of active cell division. However, in ein2-1 cells, ethylene generation was significantly reduced while NO levels were increased. With application of a range of concentrations of the NO donor, sodium nitroprusside (SNP) (between 20 and 500 µM) ethylene production was significantly diminished in Col-0 but unchanged in ein2-1 cells. Flow cytometry assays showed that in Col-0 cells treatments with 5 and 10 µM SNP concentrations led to an increase in S-phase cell number indicating the stimulation of G1/S transition. However, at ≥20 µM SNP CC progression was restrained at G1/S transition. In the mutant ein2-1 strain, the index of S-phase cells was not altered at 5-10 µM SNP but decreased dramatically at higher SNP concentrations. Concomitantly, 5 µM SNP induced transcription of genes encoding CDKA;1 and CYCD3;1 in Col-0 cells whereas transcription of CDKs and CYCs were not significantly altered in ein2-1 cells at any SNP concentrations examined. Hence, it is appears that EIN2 is required for full responses at each SNP concentration. In ein2-1 cells, greater amounts of NO, reactive oxygen species, and the tyrosine-nitrating peroxynitrite radical were detected, possibly indicating NO-dependent post-translational protein modifications which could stop CC. Thus, we suggest that in Arabidopsis cultured cells NO affects CC progression as a concentration-dependent modulator with a dependency on EIN2 for both ethylene production and a NO/ethylene regulatory function.

Founder mutations in early-onset, familial and bilateral breast cancer patients from Russia.

Previous studies indicate that founder mutations may play a noticeable role in breast cancer (BC) predisposition in Russia. Here we performed a systematic analysis of eight recurrent mutations in 302 BC cases (St.-Petersburg, Russia), which were selected due to the presence of clinical indicators of hereditary disease (bilaterality and/or early onset (< or =40 years) and/or family history). BC-associated alleles were revealed in 46 (15.2%) women. BRCA1 5382insC mutation was detected in 29 (9.6%) patients, CHEK2 1100delC in 9 (3.0%), BRCA1 4153delA in 3 (1.0%), CHEK2 IVS2+1G>A in 2 (0.7%), and BRCA1 185delAG, BRCA2 6174delT and NBS1 657del5 in 1 (0.3%) patient each. No cases with BRCA1 300T>G (C61G) mutation was identified. The obtained data suggest that a significant fraction of hereditary BC cases in Russia can be diagnosed using only a limited number of simple PCR tests.