Fabrication of organic electrochemical transistor arrays for biosensing

Background

Organic electrochemical transistors (OECT) have been used as various types of biosensors with very high sensitivity. The OECTs show advantages of easy fabrication, low operational voltage, excellent flexibility and biocompatibility.

Methods

OECT arrays based on poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) were fabricated in poly(ethylene glycol) (PEG) microwells by physical delamination.

Results

The OECTs show fast response time, stable channel current and excellent transistor characteristics. The PEG microwells can be used to trap cells on top of the OECTs, which will be important for the application of the OECT arrays as cell-based biosensors.

General significance

This technique provides a feasible way for high-throughput cell analysis based on transistor arrays. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Synthesis,copolymerization and peptide-modification of carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) for neural electrode interfaces

Background

Conjugated polymers have been developed as effective materials for interfacing prosthetic device electrodes with neural tissue. Recent focus has been on the development of conjugated polymers that contain biological components in order to improve the tissue response upon implantation of these electrodes.

Methods

Carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) monomer was synthesized in order to covalently bind peptides to the surface of conjugated polymer films. EDOTacid was copolymerized with EDOT monomer to form stable, electrically conductive copolymer films referred to as PEDOT-PEDOTacid. The peptide GGGGRGDS was bound to PEDOT-PEDOTacid to create peptide functionalized PEDOT films.

Results

The PEDOT-PEDOTacid-peptide films increased the adhesion of primary rat motor neurons between 3 and 9 times higher than controls, thus demonstrating that the peptide maintained its biological activity.

Conclusions

The EDOT-acid monomer can be used to create functionalized PEDOT-PEDOTacid copolymer films that can have controlled bioactivity.

General Significance

PEDOT-PEDOTacid-peptide films have the potential to control the behavior of neurons and vastly improve the performance of implanted electrodes. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

PEGylation of lysine residues reduces the pro-migratory activity of IGF-I

Background

The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some of these functions involve regulation by the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, the functions and exact nature of these interactions remain incomplete.

Methods

IGF-I variants PEGylated at lysines K27, K65 and K68, were assessed for binding to IGFBPs using BIAcore, and for phosphorylation of the IGF-IR. Furthermore, functional consequences of PEGylation were investigated using cell viability and migration assays. In addition, downstream signaling pathways were analyzed using phospho-AKT and phospho-ERK1/2 assays.

Results

IGF-I PEGylated at lysines 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) was employed. Receptor phosphorylation was similarly reduced 2-fold with PEG-K65 and PEG-K68 in 3T3 fibroblasts and MCF-7 breast cancer cells, whereas PEG-K27 showed a more than 10- and 3-fold lower activation for 3T3 and MCF-7 cells, respectively. In addition, all PEG-IGF-I variants had a 10-fold reduced association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes, whereas cell viability was fully preserved. Analysis of downstream signaling revealed that AKT was preferentially affected upon treatment with PEG-IGF-I variants whereas MAPK signaling was unaffected by PEGylation.

Conclusion

PEGylation of IGF-I has an impact on cell migration but not on cell viability.

General significance

PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on receptor interaction as well as key extracellular proteins such as VN and IGFBPs.     

Monitoring neural stem cell differentiation using PEDOT–PSS based MEA

Background

Transplantation is one potential clinical application of neural stem cells (NSCs). However, it is very difficult to monitor/control NSCs after transplantation and so provide effective treatment. Electrical measurement using a poly(3,4-ethylenedioxythiophene)–poly(styrenesulfonate) (PEDOT–PSS) modified microelectrode array (MEA) is a biocompatible, non-invasive, non-destructive approach to understanding cell conditions. This property makes continuous monitoring available for the evaluation/assessment of the development of cells such as NSCs.

Methods

A PEDOT–PSS modified MEA was used to monitor electrical signals during NSC development in a culture derived from rat embryo striatum in order to understand the NSC differentiation conditions.

Results

Electrical data indicated that NSCs with nerve growth factor (NGF) generate a cultured cortical neuron-like burst pattern while a random noise pattern was measured with epidermal growth factor (EGF) at 4 days in vitro (DIV) and a burst pattern was observed in both cases at 11 DIV indicating the successful monitoring of differentiation differences and developmental changes.

Conclusions

The electrical analysis of cell activity using a PEDOT–PSS modified MEA could indicate neural network formation by differentiated neurons. Changes in NSC differentiation could be monitored.

General significance

The method is based on non-invasive continuous measurement and so could prove a useful tool for the primary/preliminary evaluation of a pharmaceutical analysis. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Liposome sensing and monitoring by organic electrochemical transistors integrated in microfluidics

Background

Organic electrochemical transistors (OECTs), which are becoming more and more promising devices for applications in bioelectronics and nanomedicine, are proposed here as ideally suitable for sensing and real time monitoring of liposome-based structures. This is quite relevant since, currently, the techniques used to investigate liposomal structures, their stability in different environments as well as drug loading and delivery mechanisms, operate basically off-line and/or with pre-prepared sampling.

Methods

OECTs, based on the PEDOT:PSS conductive polymer, have been employed as sensors of liposome-based nanoparticles in electrolyte solutions to assess sensitivity and monitoring capabilities based on ion-to-electron amplified transduction.

Results

We demonstrate that OECTs are very efficient, reliable and sensitive devices for detecting liposome-based nanoparticles on a wide dynamic range down to 10^− 5 mg/ml (with a lowest detection limit, assessed in real-time monitoring, of 10^− 7 mg/ml), thus matching the needs of typical drug loading/drug delivery conditions. They are hence particularly well suited for real-time monitoring of liposomes in solution. Furthermore, OECTs are shown to sense and discriminate successive injection of different liposomes, so that they could be good candidates in quality-control assays or in the pharmaceutical industry.

General significance

Drug loading and delivery by liposome-based structures is a fast growing and very promising field that will strongly benefit from real-time, highly sensitive and low cost monitoring of their dynamics in different pharma and biomedical environments, with a particular reference to the pharmaceutical and production processes, where a major issue is monitoring and measuring the formation and concentration of liposomes and the relative drug load. The demonstrated ability to sense and monitor complex bio-structures, such as liposomes, paves the way for very promising developments in biosensing and nanomedicine. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Electrically tunable organic bioelectronics for spatial and temporal manipulation of neuron-like pheochromocytoma (PC-12) cells

Background

Organic bioelectronic devices consisting of alternating poly(3,4-ethylenedioxythiophene) (PEDOT) and reduced graphite oxide (rGO) striped microelectrode arrays were fabricated by lithography technology. It has been demonstrated that the organic bioelectronic devices can be used to spatially and temporally manipulate the location and proliferation of the neuron-like pheochromocytoma cells (PC-12 cells).

Methods

By coating an electrically labile contact repulsion layer of poly(l-lysine-graft-ethylene glycol) (PLL-g-PEG) on the PEDOT electrode, the location and polarity of the PC-12 cells were confined to the rGO electrodes.

Results

The outgrowth of spatially confined bipolar neurites was found to align along the direction of the 20 μm wide electrode. The location of the PC-12 cells can also be manipulated temporally by applying electrical stimulation during the neurite differentiation of PC-12 cells, allowing the PC-12 cells to cross over the boundary between the PEDOT and the rGO regions and construct neurite networks in an unconfined manner where the contact repulsive coating of PLL-g-PEG was removed.

Conclusions

This adsorption and desorption of the PLL-g-PEG without and with electrical stimulation can be attributed to the tunable surface properties of the PEDOT microelectrodes, whose surface charge can switch from being negative to positive under electrical stimulation.

General significance

The electrically tunable organic bioelectronics reported here could potentially be applied to tissue engineering related to the development and regeneration of mammalian nervous systems. The spatial and temporal control in this device would also be used to study the synapse junctions of neuron–neuron contacts in both time and space domains. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated chitosan nanoparticles towards colon cancer treatment

Background

Evaluation of the combinatorial anticancer effects of curcumin/5-fluorouracil loaded thiolated chitosan nanoparticles (CRC-TCS-NPs/5-FU-TCS-NPs) on colon cancer cells and the analysis of pharmacokinetics and biodistribution of CRC-TCS-NPs/5-FU-TCS-NPs in a mouse model.

Methods

CRC-TCS-NPs/5-FU-TCS-NPs were developed by ionic cross-linking. The in vitro combinatorial anticancer effect of the nanomedicine was proven by different assays. Further the pharmacokinetics and biodistribution analyses were performed in Swiss Albino mouse using HPLC.

Results

The 5-FU-TCS-NPs (size: 150 ± 40 nm, zeta potential: + 48.2 ± 5 mV) and CRC-TCS-NPs (size: 150 ± 20 nm, zeta potential: + 35.7 ± 3 mV) were proven to be compatible with blood. The in vitro drug release studies at pH 4.5 and 7.4 showed a sustained release profile over a period of 4 days, where both the systems exhibited a higher release in acidic pH. The in vitro combinatorial anticancer effects in colon cancer (HT29) cells using MTT, live/dead, mitochondrial membrane potential and cell cycle analysis measurements confirmed the enhanced anticancer effects (2.5 to 3 fold). The pharmacokinetic studies confirmed the improved plasma concentrations of 5-FU and CRC up to 72 h, unlike bare CRC and 5-FU.

Conclusions

To conclude, the combination of 5-FU-TCS-NPs and CRC-TCS-NPs showed enhanced anticancer effects on colon cancer cells in vitro and improved the bioavailability of the drugs in vivo.

General significance

The enhanced anticancer effects of combinatorial nanomedicine are advantageous in terms of reduction in the dosage of 5-FU, thereby improving the chemotherapeutic efficacy and patient compliance of colorectal cancer cases.     

Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells

Background

Changes in fibronectin (Fn) matrix remodeling contribute to mammary tumor angiogenesis and are related to altered behavior of adipogenic stromal cells; yet, the underlying mechanisms remain unclear due in part to a lack of reductionist model systems that allow the inherent complexity of cell-derived extracellular matrices (ECMs) to be deciphered. In particular, breast cancer-associated adipogenic stromal cells not only enhance the composition, quantity, and rigidity of deposited Fn, but also partially unfold these matrices. However, the specific effect of Fn conformation on tumor angiogenesis is undefined.

Methods

Decellularized matrices and a conducting polymer device consisting of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) were used to examine the effect of Fn conformation on the behavior of 3T3-L1 preadipocytes. Changes in cell adhesion and proangiogenic capability were tested via cell counting and by quantification of vascular endothelial growth factor (VEGF) secretion, respectively. Integrin-blocking antibodies were utilized to examine varied integrin specificity as a potential mechanism.

Results

Our findings suggest that tumor-associated partial unfolding of Fn decreases adhesion while enhancing VEGF secretion by breast cancer-associated adipogenic precursor cells, and that altered integrin specificity may underlie these changes.

Conclusions and general significance

These results not only have important implications for our understanding of tumorigenesis, but also enhance knowledge of cell-ECM interactions that may be harnessed for other applications including advanced tissue engineering approaches. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

Extracellular matrix: A dynamic microenvironment for stem cell niche

Background

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche.

Scope of review

We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions.

Major conclusions

ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior.

General significance

ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

Background

Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.

Methods

Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.

Results

The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.

Conclusions

These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.

General significance

This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.     

Matrikines from basement membrane collagens: A new anti-cancer strategy

Background

Tumor microenvironment is a complex system composed of a largely altered extracellular matrix with different cell types that determine angiogenic responses and tumor progression. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. In addition to stromal ECM breakdown, proteases exert various pro- or anti-tumorigenic functions and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to act as endogenous angiogenesis inhibitors and to limit tumor progression.

Scope of review

We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV.

Major conclusions

The putative targets of the matrikine control are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. Collagen-derived matrikines such as canstatin, tumstatin or tetrastatin for example, decrease tumor growth in various cancer models. Their anti-cancer activities comprise anti-proliferative effects on tumor or endothelial cells by induction of apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype. They were used in various preclinical therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogues designed from the structure of the active sequences, iii) used in combined therapies with conventional chemotherapy or radiotherapy.

General significance

Collagen-derived matrikines strongly inhibited tumor growth in many preclinical cancer models in mouse. They constitute a new family of anti-cancer agents able to limit cancer progression. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Alteration of cell membrane proteoglycans impairs FSH receptor/Gs coupling and ERK activation through PP2A-dependent mechanisms in immature rat Sertoli cells

Background

During the pre-pubertal life, the cessation of Sertoli cell proliferation and the onset of differentiation are associated with a shift in the FSH-mediated signaling leading to inhibition of the ERK-mitogenic pathway and to a concomitant sensitization of cAMP/PKA pathway.

Methods

To highlight the role of cell proteoglycans (PGs) in the shift of FSH signaling, both FSH-induced cAMP production and ERK1/2 inactivation were studied in untreated and sodium chlorate PG-depleted cultured Sertoli cells from 20 day-old rats.

Results

Depletion of cell membrane PGs by sodium chlorate reduced FSH-, but not cholera toxin-stimulated cAMP production as well as basal ERK phosphorylation through an okadaic acid (OA)-sensitive mechanism. Involvement of PP2A was further substantiated by a marked decrease in membrane- associated PP2A activity under SC conditions and by the OA-induced restoration of PKA-dependent ERK inactivation in SC-treated cells.

Conclusions

In 20-day-old rat Sertoli cells, transmembrane cell PGs, through tethering/activation of PP2A activity exerts regulatory control on both FSH receptor/Gs coupling and ERK phosphorylation.

General significance

Besides their antiproliferative roles, cell PGs such as syndecan-1, could be involved in the increase in cAMP response to FSH occurring in Sertoli cells at the time of transition between proliferative and differentiated states.     

Organic bioelectronics for electronic-to-chemical translation in modulation of neuronal signaling and machine-to-brain interfacing

Background

A major challenge when creating interfaces for the nervous system is to translate between the signal carriers of the nervous system (ions and neurotransmitters) and those of conventional electronics (electrons).

Scope of review

Organic conjugated polymers represent a unique class of materials that utilizes both electrons and ions as charge carriers. Based on these materials, we have established a series of novel communication interfaces between electronic components and biological systems. The organic electronic ion pump (OEIP) presented in this review is made of the polymer–polyelectrolyte system poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). The OEIP translates electronic signals into electrophoretic migration of ions and neurotransmitters.

Major conclusions

We demonstrate how spatio-temporally controlled delivery of ions and neurotransmitters can be used to modulate intracellular Ca^2 + signaling in neuronal cells in the absence of convective disturbances. The electronic control of delivery enables strict control of dynamic parameters, such as amplitude and frequency of Ca^2 + responses, and can be used to generate temporal patterns mimicking naturally occurring Ca^2 + oscillations. To enable further control of the ionic signals we developed the electrophoretic chemical transistor, an analog of the traditional transistor used to amplify and/or switch electronic signals. Finally, we demonstrate the use of the OEIP in a new “machine-to-brain” interface by modulating brainstem responses in vivo.

General significance

This review highlights the potential of communication interfaces based on conjugated polymers in generating complex, high-resolution, signal patterns to control cell physiology. We foresee widespread applications for these devices in biomedical research and in future medical devices within multiple therapeutic areas. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: comparison with influence of poly(sulfoanions)

Background

It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Methods

Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein–polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity.

Results

Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH.

Conclusions

The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications.

General significance

Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.     

Multi-target-directed design,syntheses, and characterization of fluorescent bisphosphonate derivatives as multifunctional enzyme inhibitors in mevalonate pathway

Background

Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. Four enzymes in mevalonate pathway, including MVK, PMK, MDD, and FPPS, play important regulatory roles in cholesterol biosynthesis and cell proliferation.

Methods

The following methods were used: cloning, expression and purification of enzymes in mevalonate pathway, organic syntheses of multifunctional enzyme inhibitors, measurement of their IC₅₀ values for above four enzymes, kinetic studies of enzyme inhibitions, molecular modeling studies, cell viability tests, and fluorescence microscopy.

Results and conclusions

We report our multi-target-directed design, syntheses, and characterization of two blue fluorescent bisphosphonate derivatives compounds 15 and 16 as multifunctional enzyme inhibitors in mevalonate pathway. These two compounds had good inhibition to all these four enzymes with their IC₅₀ values at nanomolar to micromolar range. Kinetic and molecular modeling studies showed that these two compounds could bind to the active sites of all these four enzymes. The fluorescence microscopy indicated that these two compounds could easily get into cancer cells.

General significance

Multifunctional enzyme inhibitors are generally more effective than single enzyme inhibitors, with fewer side effects. Our results showed that these multifunctional inhibitors could become lead compounds for further development for the treatment of soft-tissue tumors and hypercholesteremia.     

Modulation of Dcytb (Cybrd 1) expression and function by iron,dehydroascorbate and Hif-2α in cultured cells

Background

Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines.

Methods

Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and ⁵⁹Fe uptake assays.

Results

Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake.

Conclusions

Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α.

General significance

Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes.     

Loss of HuR leads to senescence-like cytokine induction in rodent fibroblasts by activating NF-κB

Background

HuR (human antigen R) is a ubiquitously expressed member of the Hu/ELAV family of proteins that is involved in diverse biological processes. HuR has also been shown to play an important role in cell cycle arrest during replicative senescence in both human and mouse cells. Senescent cells not only halt their proliferation, but also activate the secretion of proinflammatory cytokines. A persistent DNA damage response is essential for the senescence-associated secretory phenotype (SASP), and increasing evidence has suggested that the SASP is associated with malignancy.

Methods

Senescence-associated phenotypes were analyzed in MEFs and other cell line in which HuR expression is inhibited by sh-RNA-mediated knockdown.

Results

RNAi-mediated HuR inhibition resulted in an increase in SASP-related cytokines. The induction of SASP factors did not depend on ARF–p53 pathway-mediated cell cycle arrest, but required NF-κB activity. In the absence of HuR, cells were defective in the DNA-damage response, and single strand DNA breaks accumulated, which may have caused the activation of NF-κB and subsequent cytokine induction.

Conclusions

In the absence of HuR, cells exhibit multiple senescence-associated phenotypes. Our findings suggest that HuR regulates not only the replicative lifespan, but also the expression of SASP-related cytokines in mouse fibroblasts.

General significance

RNA-binding protein HuR protects cells from undergoing senescence. Senescence-associated phenotypes are accelerated in HuR-deficient cells.     

Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation

Background

Stem cell therapy is a strategy far from being satisfactory and applied in the clinic. Poor survival and differentiation levels of stem cells after transplantation or neural injury have been major problems. Recently, it has been recognized that cell death-relevant proteins, notably those that operate in the core of the executioner apoptosis machinery are functionally involved in differentiation of a wide range of cell types, including neural cells.

Scope of review

This article will review recent studies on the mechanisms underlying the non-apoptotic function of mitochondrial and death receptor signaling pathways during neural differentiation. In addition, we will discuss how these major apoptosis-regulatory pathways control the decision between differentiation, self-renewal and cell death in neural stem cells and how levels of activity are restrained to prevent cell loss as final outcome.

Major conclusions

Emerging evidence suggests that, much like p53, caspases and Bcl-2 family members, the two prime triggers of cell death pathways, death receptors and mitochondria, may influence proliferation and differentiation potential of stem cells, neuronal plasticity, and astrocytic versus neuronal stem cell fate decision.

General significance

A better understanding of the molecular mechanisms underlying key checkpoints responsible for neural differentiation as an alternative to cell death will surely contribute to improve neuro-replacement strategies.     

Validation of the organic electrochemical transistor for in vitro toxicology

Background

The gastrointestinal epithelium provides a physical and biochemical barrier to the passage of ions and small molecules; however this barrier may be breached by pathogens and toxins. The effect of individual pathogens/toxins on the intestinal epithelium has been well characterized: they disrupt barrier tissue in a variety of ways, such as by targeting tight junction proteins, or other elements of the junctions between adjacent cells. A variety of methods have been used to characterize disruption in barrier tissue, such as immunofluorescence, permeability assays and electrical measurements of epithelia resistance, but these methods remain time consuming, costly and ill-suited to diagnostics or high throughput toxicology.

Methods

The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the organic electrochemical transistor (OECT), whose low cost materials and potential for easy fabrication in high throughput formats represent a novel solution for assessing epithelial tissue integrity.

Results

In this study, OECTs were integrated with gastro-intestinal cell monolayers to study the integrity of the gastrointestinal epithelium, providing a very sensitive way to detect minute changes in ion flow across the cell layer due to inherent amplification by the transistor.

Major conclusions

We validate the OECT against traditional methods by monitoring the effect of toxic compounds on epithelial tissue. We show a systematic characterization of this novel method, alongside existing methods used to assess barrier tissue function.

General significance

The toxic compounds induce a dramatic disruption of barrier tissue, and the OECT measures this disruption with increased temporal resolution and greater or equal sensitivity when compared with existing methods. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.