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The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

NAD kinase is the sole NADP(+) biosynthetic enzyme. Despite the great significance of NADP(+), to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP(+) remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP(+). The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP(+) biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP(+) biosynthesis, which has pivotal roles in cells and organelles.

Although the key factor affecting the biocompatibility of IONPs is the core size, there is a lack of regular investigation concerning the impact of the parameter on the toxicity of these nanomaterials. Therefore, such studies were carried out in this paper. Their purpose was to compare the influence of PEG-coated-magnetite NPs with the core of 5, 10 and 30 nm on six carefully selected cell lines. The proliferation rate, viability, metabolic activity, migration activity, ROS levels and cytoskeleton architecture of cells have been evaluated for specified incubation periods. These were 24 and 72-h long incubations with IONPs administered in two doses: 5 and 25 µg Fe/ml. A decrease in viability was observed after exposure to the tested NPs for all the analyzed cell lines. This effect was not connected with core diameter but depended on the exposure time to the nanomaterials. IONPs increased not only the proliferation rate of macrophages-being phagocytic cells-but also, under certain conditions stimulated tumor cell divisions. Most likely, the increase in proliferation rate of macrophages contributed to the changes in the architecture of their cytoskeleton. The growth in the level of ROS in cells had been induced mainly by the smallest NPs. This effect was observed for HEK293T cells and two cancerous lines: U87MG (at both doses tested) and T98G (only for the higher dose). This requires further study concerning both potential toxicity of such IONPs to the kidneys and assessing their therapeutic potential in the treatment of glioblastoma multiforme.

The application of antioxidants has been considered as an important and effective approach against conditions in which oxidative stress occurs. Especially, ferulic acid (FA) is an important antioxidant which exerts potency against cellular damage in the presence of oxidants. In the current study, the resistance effect of FA on hydrogen peroxide (H₂O₂)-stressed human embryonic kidney 293 cells (HEK293) in vitro was investigated. FA (1 mM) increased HEK293 cells’ viability and significantly reduced H₂O₂-induced cellular apoptosis, which was confirmed with flow cytometry and morphological results. Cell cycle analysis indicated low percentage of sub-G₀ population of FA-treated HEK293 cells that confirmed its resistance effect. The FA-treated HEK293 cells followed by H₂O₂ exposure resulted in decreased ROS levels compared to control (H₂O₂-treated only). The results indicated that pretreatment of FA on cell prior to H₂O₂ exposure could significantly improve cell survival and increase catalase (CAT) and superoxide dismutase (SOD) levels. On the other hand, reduction in the levels of MDA and ROS was obvious. It can be concluded that FA may protect HEK293 cells from injury induced by H₂O₂ through regulation of intracellular antioxidant enzyme activities and cell cycle distribution. The reduction in mitochondrial membrane potential was also inhibited by FA treatment. These results suggested the importance of naturally occurring antioxidants such as FA in therapeutic intervention methodology against oxidative stress-related diseases.

Exogenous nutrient elements modulate the energetic metabolism responses that are prerequisites for cellular homeostasis and metabolic physiology. Although zinc is important in oxidative stress and cytoprotection processes, its role in the regulation of energetic metabolism remains largely unknown. In this study, we found that zinc stimulated aspect in cell motility and was essential in restoring the Ochratoxin A (OTA)-induced energetic metabolism damage in HEK293 cells. Moreover, using zinc supplementation and zinc deficiency models, we observed that zinc is conducive to mitochondrial pyruvate transport, oxidative phosphorylation, carbohydrate metabolism, lipid metabolism and ultimate energy metabolism in both normal and toxic-induced oxidative stress conditions in vitro, and it plays an important role in restoring impaired energetic metabolism. This zinc-mediated energetic metabolism regulation could also be helpful for DNA maintenance, cytoprotection and hereditary cancer traceability. Therefore, zinc can widely adjust energetic metabolism and is essential in restoring the impaired energetic metabolism of cellular physiology.

The HIV-1 accessory protein Vpu counteracts tetherin (BST-2/CD317) by preventing its incorporation into virions, reducing its surface expression, and ultimately promoting its degradation. Here we characterize a putative trafficking motif, EXXXLV, in the second alpha helix of the subtype-B Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents ESCRT-dependent degradation of tetherin/Vpu complexes, tetherin cell surface downregulation, but not its physical interaction with Vpu. Importantly, this motif is required for efficient cell-free virion release from CD4+ T cells, particularly after their exposure to type-1 interferon, indicating that the ability to reduce surface tetherin levels and promote its degradation is important to counteract restriction under conditions that the virus likely encounters in vivo. Vpu EXXXLV mutants accumulate with tetherin at the cell surface and in endosomal compartments, but retain the ability to bind both β-TrCP2 and HRS, indicating that this motif is required for a post-binding trafficking event that commits tetherin for ESCRT-dependent degradation and prevents its transit to the plasma membrane and viral budding zones. We further found that while Vpu function is dependent on clathrin, and the entire second alpha helix of the Vpu tail can be functionally complemented by a clathrin adaptor binding peptide derived from HIV-1 Nef, none of the canonical clathrin adaptors nor retromer are required for this process. Finally we show that residual activity of Vpu EXXXLV mutants requires an intact endocytic motif in tetherin, suggesting that physical association of Vpu with tetherin during its recycling may be sufficient to compromise tetherin activity to some degree.

Epidemiological case-control studies have revealed associations between mitochondrial haplogroups and the onset and/or progression of various multifactorial diseases. For instance, mitochondrial haplogroup T was previously shown to be associated with vascular diseases, including coronary artery disease and diabetic retinopathy. In contrast, haplogroup H, the most frequent haplogroup in Europe, is often found to be more prevalent in healthy control subjects than in patient study groups. However, justifications for the assumption that haplogroups are functionally distinct are rare. Therefore, we attempted to compare differences in mitochondrial function between haplogroup H and T cybrids. Mitochondrial haplogroup H and T cybrids were generated by fusion of HEK293 cells devoid of mitochondrial DNA with isolated thrombocytes of individuals with the respective haplogroups. These cybrid cells were analyzed for oxidative phosphorylation (OXPHOS) enzyme activities, mitochondrial DNA (mtDNA) copy number, growth rate and susceptibility to reactive oxygen species (ROS). We observed that haplogroup T cybrids have higher survival rate when challenged with hydrogen peroxide, indicating a higher capability to cope with oxidative stress. The results of this study show that functional differences exist between HEK293 cybrid cells which differ in mitochondrial genomic background.

Influenza A virus (IAV) belongs to the family. IAV causes a highly contagious respiratory disease in humans that exacts severe economic losses globally. The virus uses strategies developed to exploit and subvert cellular proteins and pathways to increase its own replication and to inhibit antiviral immune response. A/bar-headed goose/Qinghai/1/2005 (A/QH) was able to infect A549 and 293 T cells, with a high infection rate for A549 cells. To identify host cellular responses of human cells to influenza infection, differentially expressed genes (DEGs) between AIV-infected groups and uninfected controls were identified using RNA-sequencing. The DEGs were annotated by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses, which revealed that the DEGs were mainly linked to cellular function and metabolic processes, while the cellular function that is probably associated with host cellular response of human cells, including defense response to virus and protein modification. All the DEGs and pathways were possibly involved in the response to IAV invasion. The global transcriptome analysis results revealed that sensitive genes and pathways of the cells were infected with the influenza virus and provided further evidence to investigate the complicated relationship between IAV and host cells.

Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K(+) channels expressed in HEK293 cells. hERG K(+) currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K(+) channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy. Application of ATV (0.01-30 μmol/L) concentration-dependently decreased hERG K(+) currents with an IC50 of 5.7±1.8 μmol/L. ATV (10 μmol/L) did not affect the activation and steady-state inactivation of hERG K(+) currents. Compared with the wild type hERG K(+) channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K(+) currents. Overnight treatment with ATV (0.1-30 μmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane. ATV directly blocks hERG K(+) channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.