RNA-binding proteins regulating the CD44 alternative splicing

Alternative splicing is often deregulated in cancer, and cancer-specific isoform switches are part of the oncogenic transformation of cells. Accumulating evidence indicates that isoforms of the multifunctional cell-surface glycoprotein CD44 play different roles in cancer cells as compared to normal cells. In particular, the shift of CD44 isoforms is required for epithelial to mesenchymal transition (EMT) and is crucial for the maintenance of pluripotency in normal human cells and the acquisition of cancer stem cells phenotype for malignant cells. The growing and seemingly promising use of splicing inhibitors for treating cancer and other pathologies gives hope for the prospect of using such an approach to regulate CD44 alternative splicing. This review integrates current knowledge about regulating CD44 alternative splicing by RNA-binding proteins

Transcriptome of Extracellular Vesicles: State-of-the-Art

Exosomes and microvesicles are two major categories of extracellular vesicles (EVs) released by almost all cell types and are highly abundant in biological fluids. Both the molecular composition of EVs and their release are thought to be strictly regulated by external stimuli. Multiple studies have consistently demonstrated that EVs transfer proteins, lipids and RNA between various cell types, thus mediating intercellular communication, and signaling. Importantly, small non-coding RNAs within EVs are thought to be major contributors to the molecular events occurring in the recipient cell. Furthermore, RNA cargo in exosomes and microvesicles could hold tremendous potential as non-invasive biomarkers for multiple disorders, including pathologies of the immune system. This mini-review is aimed to provide the state-of-the-art in the EVs-associated RNA transcriptome field, as well as the comprehensive analysis of previous studies characterizing RNA content within EVs released by various cells using next-generation sequencing. Finally, we highlight the technical challenges associated with obtaining pure EVs and deep sequencing of the EV-associated RNAs

Chip-based liver equivalents for toxicity testing - organotypicalness versus cost-efficient high throughput

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Drug-induced liver toxicity dominates the reasons for pharmaceutical product ban, withdrawal or non-approval since the thalidomide disaster in the late-1950s. Hopes to finally solve the liver toxicity test dilemma have recently risen to a historic level based on the latest progress in human microfluidic tissue culture devices. Chip-based human liver equivalents are envisaged to identify liver toxic agents regularly undiscovered by current test procedures at industrial throughput. In this review, we focus on advanced microfluidic microscale liver equivalents, appraising them against the level of architectural and, consequently, functional identity with their human counterpart in vivo. We emphasise the inherent relationship between human liver architecture and its drug-induced injury. Furthermore, we plot the current socio-economic drug development environment against the possible value such systems may add. Finally, we try to sketch a forecast for translational innovations in the field

Fluorescent optical fiber sensors for cell viability monitoring

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.A new simple method for non-invasive cell culture viability monitoring based on vital fluorescent stains is introduced, and its efficiency for long-term experiments on cells is demonstrated. In contrast to common methods for cell viability control, which are usually either destructive (like flow-type counters or dead cells coloring and counting), or hardly quantitative like fluorescent microscopy, the method described is automated, does not require the removal of cells from their growth area and is sensitive enough to deal with as low as tens of cells

A new approach to studies of cell-antibody interactions using fluorescent lipid probes

AbstractThe interaction of poly- and monoclonal antibodies against the L-chain of human Ig with Burkitt lymphoma EB-3 cells was studied using a fluorescent lipid probe, anthrylvinyl-labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell. The possibility of using cells or liposomes carrying antigens and fluorescent lipids for the determination of antibodies in solution is discussed

Membrane fusion mediated by ricin and viscumin

AbstractThe ribosome inactivating plant proteins (RIPs) ricin and viscumin but not Ricinus communis agglutinin are able induce vesicle–vesicle fusion. A model is suggested in which the toxicity of the RIPs is partially determined by their fusogenicity. Herein, fusion is hypothesized to allow the RIPs to leak across endocytic vesicles to approve their access to cytoplasmic ribosomes

In Vitro and in Silico Liver Models: Current Trends, Challenges and Opportunities

Most common drug development failures originate from either bioavailability problems, or unexpected toxic effects. The culprit is often the liver, which is responsible for biotransformation of a majority of xenobiotics. Liver may be modeled using liver on a chip devices, which may include established cell lines, primary human cells, and stem cell-derived hepatocyte-like cells. The choice of biological material along with its processing and maintenance greatly influence both the device performance and the resultant toxicity predictions. Impediments to the development of liver on a chip technology include the problems with standardization of cells, limitations imposed by culturing and the necessity to develop more complicated fluidic contours. Fortunately, recent breakthroughs in the development of cell-based reporters, including ones with fluorescent label, permits monitoring of the behavior of the cells embed into the liver on a chip devices. Finally, a set of computational approaches has been developed to model both particular toxic response and the homeostasis of human liver as a whole; these approaches pave a way to enhance the in silico stage of assessment for a potential toxicity

Torque Teno Virus (TTV) distribution in healthy Russian population

<p>Abstract</p> <p>Background</p> <p>Torque teno virus (TTV) is a circular, single-stranded DNA virus that chronically infects healthy individuals of all ages worldwide. There is a lot of data on the prevalence and genetic heterogeneity of TTV in healthy populations and in patients with various diseases now available. However, little is known about TTV load among healthy human population. In this study we analyzed TTV load in the group of 512 Russian elite athletes, who are supposed to be, by some standards, the healthiest part of the human population.</p> <p>Results</p> <p>The prevalence rate of TTV among the Russian Olympic Reserve members was 94% (for test sensitivity about 1000 genome equivalents per 1 ml of blood). Quantities varied from 10³(which corresponded to detection limit) to 10¹⁰copies per 1 ml of blood, with median at 2.7 × 10⁶copies.</p> <p>Conclusion</p> <p>About 94% of healthy individuals in Russian population have more than 1000 TTV genome copies per 1 ml of blood. This result exceeds the previously published data, and can be explained by either more sensitive PCR test system or by higher TTV distribution in Russian population or both. TTV viral load neither depends on gender, nor age.</p

Mistletoe lectin dissociates into catalytic and binding subunits before translocation across the membrane to the cytoplasm

AbstractHybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A-chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome-inactivating proteins. Anti-MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin