Altered Mitochondrial Morphology and Bioenergetics in a New Yeast Model Expressing Aß42.

Alzheimer's disease (AD) is an incurable, age-related neurological disorder, the most common form of dementia. Considering that AD is a multifactorial complex disease, simplified experimental models are required for its analysis. For this purpose, genetically modified Yarrowia lipolytica yeast strains expressing Aß42 (the main biomarker of AD), eGFP-Aß42, Aß40, and eGFP-Aß40 were constructed and examined. In contrast to the cells expressing eGFP and eGFP-Aß40, retaining "normal" mitochondrial reticulum, eGFP-Aß42 cells possessed a disturbed mitochondrial reticulum with fragmented mitochondria; this was partially restored by preincubation with a mitochondria-targeted antioxidant SkQThy. Aß42 expression also elevated ROS production and cell death; low concentrations of SkQThy mitigated these effects. Aß42 expression caused mitochondrial dysfunction as inferred from a loose coupling of respiration and phosphorylation, the decreased level of ATP production, and the enhanced rate of hydrogen peroxide formation. Therefore, we have obtained the same results described for other AD models. Based on an analysis of these and earlier data, we suggest that the mitochondrial fragmentation might be a biomarker of the earliest preclinical stage of AD with an effective therapy based on mitochondria- targeted antioxidants. The simple yeast model constructed can be a useful platform for the rapid screening of such compounds.

The First Yarrowia lipolytica Yeast Models Expressing Hepatitis B Virus X Protein: Changes in Mitochondrial Morphology and Functions.

Chronic hepatitis B virus infection is the dominant cause of hepatocellular carcinoma, the main cause of cancer death. HBx protein, a multifunctional protein, is essential for pathogenesis development; however, the underlying mechanisms are not fully understood. The complexity of the system itself, and the intricate interplay of many factors make it difficult to advance in understanding the mechanisms underlying these processes. The most obvious solution is to use simpler systems by reducing the number of interacting factors. Yeast cells are particularly suitable for studying the relationships between oxidative stress, mitochondrial dynamics (mitochondrial fusion and fragmentation), and mitochondrial dysfunction involved in HBx-mediated pathogenesis. For the first time, genetically modified yeast, Y. lipolytica, was created, expressing the hepatitis B virus core protein HBx, as well as a variant fused with eGFP at the C-end. It was found that cells expressing HBx experienced stronger oxidative stress than the control cells. Oxidative stress was alleviated by preincubation with the mitochondria-targeted antioxidant SkQThy. Consistent with these data, in contrast to the control cells (pZ-0) containing numerous mitochondrial forming a mitochondrial reticulum, in cells expressing HBx protein, mitochondria were fragmented, and preincubation with SkQThy partially restored the mitochondrial reticulum. Expression of HBx had a significant influence on the bioenergetic function of mitochondria, making them loosely coupled with decreased respiratory rate and reduced ATP formation. In sum, the first highly promising yeast model for studying the impact of HBx on bioenergy, redox-state, and dynamics of mitochondria in the cell and cross-talk between these parameters was offered. This fairly simple model can be used as a platform for rapid screening of potential therapeutic agents, mitigating the harmful effects of HBx.

Lamin A as a Determinant of Mechanical Properties of the Cell Nucleus in Health and Disease.

One of the main factors associated with worse prognosis in oncology is metastasis, which is based on the ability of tumor cells to migrate from the primary source and to form secondary tumors. The search for new strategies to control migration of metastatic cells is one of the urgent issues in biomedicine. One of the strategies to stop spread of cancer cells could be regulation of the nuclear elasticity. Nucleus, as the biggest and stiffest cellular compartment, determines mechanical properties of the cell as a whole, and, hence, could prevent cell migration through the three-dimensional extracellular matrix. Nuclear rigidity is maintained by the nuclear lamina, two-dimensional network of intermediate filaments in the inner nuclear membrane (INM). Here we present the most significant factors defining nucleus rigidity, discuss the role of nuclear envelope composition in the cell migration, as well consider possible approaches to control lamina composition in order to change plasticity of the cell nucleus and ability of the tumor cells to metastasize.

Mapping mechanical properties of living cells at nanoscale using intrinsic nanopipette-sample force interactions.

Mechanical properties of living cells determined by cytoskeletal elements play a crucial role in a wide range of biological functions. However, low-stress mapping of mechanical properties with nanoscale resolution but with a minimal effect on the fragile structure of cells remains difficult. Scanning Ion-Conductance Microscopy (SICM) for quantitative nanomechanical mapping (QNM) is based on intrinsic force interactions between nanopipettes and samples and has been previously suggested as a promising alternative to conventional techniques. In this work, we have provided an alternative estimation of intrinsic force and stress and demonstrated the possibility to perform qualitative and quantitative analysis of cell nanomechanical properties of a variety of living cells. Force estimation on decane droplets with well-known elastic properties, similar to living cells, revealed that the forces applied using a nanopipette are much smaller than in the case using atomic force microscopy. We have shown that we can perform nanoscale topography and QNM using a scanning procedure with no detectable effect on live cells, allowing long-term QNM as well as detection of nanomechanical properties under drug-induced alterations of actin filaments and microtubulin.

Thiourea Modified Doxorubicin: A Perspective pH-Sensitive Prodrug.

A novel approach to the synthesis of pH-sensitive prodrugs has been proposed: thiourea drug modification. Resulting prodrugs can release the cytotoxic agent and the biologically active 2-thiohydantoin in the acidic environment of tumor cells. The concept of acid-catalyzed cyclization of thioureas to 2-thiohydantoins has been proven using a FRET model. Dual prodrugs of model azidothymidine, cytotoxic doxorubicin, and 2-thiohydantoin albutoin were obtained, which release the corresponding drugs in the acidic environment. The resulting doxorubicin prodrug was tested on prostate cancer cells and showed that the thiourea-modified prodrug is less cytotoxic (average IC50 ranging from 0.5584 to 0.9885 µM) than doxorubicin (IC50 ranging from 0.01258 to 0.02559 µM) in neutral pH 7.6 and has similar toxicity (average IC50 ranging from 0.4970 to 0.7994 µM) to doxorubicin (IC50 ranging from 0.2303 to 0.8110 µM) under mildly acidic conditions of cancer cells. Cellular and nuclear accumulation in PC3 tumor cells of Dox prodrug is much higher than accumulation of free doxorubicin.