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Cell death plays a major role in development, pathology and aging and can be triggered by various types of acute external and internal stimuli, such as chemicals or mutations. However, little is known about chronic cell death in the context of continuing cell division. Here, we performed a genome-wide search for mutants with this type of death in dividing bakers yeast by assaying the accumulation of phloxine B, which stains dead cells. We identified 83 essential and 43 non-essential gene mutants. Surprisingly, three distinct types of spatial distribution of dead cells in colonies were observed which corresponded to gene ontology enrichments for (i) DNA replication and repair, RNA processing, chromatin organization, and nuclear transport; (ii) mitosis and cytokinesis; and (iii) vesicular transport and glycosylation/cell wall homeostasis. We further developed methods for analyzing the death of newborn cells (DON) and cell death in real time using microfluidics-based microscopy which revealed rapid stochastic necrosis during bud generation or cytokinesis without prior division arrest. This coincided with commonality of sensitivity to some plasma membrane and cell-wall perturbing agents, as well as mitigating effects of increasing external pH for most of the tested mutants. Our results suggest that rapid stochastic necrosis during cell division is a common type of cell death resulting from the dysfunction of different genes, and that this type of death seems to have a common proximal cause which might be related to the properties of the cell wall and/or plasma membrane.

To develop a simple robust methodology of screening multiple CHO cell clones secreting recombinant proteins to assess their specific productivity. 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. 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.

Proteins of the AID/APOBEC family are capable of cytidine deamination in nucleic acids forming uracil. These enzymes are involved in mRNA editing, protection against viruses, the introduction of point mutations into DNA during somatic hypermutation, and antibody isotype switching. Since these deaminases, especially AID, are potent mutagens, their expression, activity, and specificity are regulated by several intra-cellular mechanisms. In this review, we discuss the mechanisms of impaired expression and activation of AID/APOBEC proteins in human tumors and their role in carcinogenesis and tumor progression. Also, the diagnostic and potential therapeutic value of increased expression of AID/APOBEC in different types of tumors is analyzed. We assume that in the case of solid tumors, increased expression of endogenous deaminases can serve as a marker of response to immunotherapy since multiple point mutations in host DNA could lead to amino acid substitutions in tumor proteins and thereby increase the frequency of neoepitopes.

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.

HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.

Cytochrome c is a well-known mitochondrial protein that fulfills life-supporting functions by transferring electrons to the respiratory chain to maintain ATP production. However, during the activation of apoptotic machinery, it is released from mitochondria and, being in the cytosol, it either triggers the activation of the caspase cascade in intrinsic apoptotic pathway, or it is involved in the amplification of extrinsic apoptotic signaling. Accumulating evidence suggests that only unmodified holocytochrome c is efficient in the stimulation of apoptosis. Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the complete or partial cytochrome c deficiency in vivo. Here, we discuss the importance of distinct amino acid residues for various functions of cytochrome c in cells and mice with targeted cytochrome c mutations.