Energy harvesting from plants using hybrid microbial fuel cells; potential applications and future exploitation.

Microbial Fuel Cells (MFC) can be fuelled using biomass derived from dead plant material and can operate on plant produced chemicals such as sugars, carbohydrates, polysaccharides and cellulose, as well as being "fed" on a regular diet of primary biomass from plants or algae. An even closer relationship can exist if algae (e.g., prokaryotic microalgae or eukaryotic and unicellular algae) can colonise the open to air cathode chambers of MFCs driving photosynthesis, producing a high redox gradient due to the oxygenic phase of collective algal cells. The hybrid system is symbiotic; the conditions within the cathodic chamber favour the growth of microalgae whilst the increased redox and production of oxygen by the algae, favour a more powerful cathode giving a higher maximum voltage and power to the photo-microbial fuel cell, which can ultimately be harvested for a range of end-user applications. MFCs can utilise a wide range of plant derived materials including detritus, plant composts, rhizodeposits, root exudates, dead or dying macro- or microalgae, via Soil-based Microbial Fuel Cells, Sediment Microbial Fuel Cells, Plant-based microbial fuel cells, floating artificial islands and constructed artificial wetlands. This review provides a perspective on this aspect of the technology as yet another attribute of the benevolent Bioelectrochemical Systems.

Vascular endothelial growth factor (VEGF) -A, -C and VE-cadherin as potential biomarkers in early breast cancer patients.

BACKGROUND: Vascular endothelial growth factor (VEGF) -A and -C act as multifunctional molecules and growth factors, while VE-cadherin (cadherin 5, CDH5) is the endothelial junction protein. AIM: To assess the relationship between intratumoral VEGF -A, -C and CDH5 levels and clinical outcome, in primary, early-stage, breast cancer patients. PATIENTS AND METHODS: The study included 69 node-negative (N0) breast cancer patients, all of whom had not received any prior hormonal or chemotherapeutic systemic therapy that would affect the course of disease. The median follow-up period was 144 months. Intratumoral mRNA levels of VEGF -A, -C and CDH5 were determined by RT-qPCR. Prognostic performance was evaluated by Cox proportional hazards regression, Kaplan-Meier analysis, as well as by the multivariable approach based on the least absolute shrinkage and selection operator (LASSO) logit regression. Classification of patients into the low and high subgroups was performed using the outcome-oriented cut-off point categorization approach. RESULTS: Of the measured mRNAs, only CDH5 mRNA (t = -2.17; p = 0.04) and VEGF-C mRNA (t = -2.41; p = 0.03) showed significant differences between values in patient subgroups with distant metastasis and those without recurrences, respectively. These t-test results were in agreement with the Cox regression by which CDH5 mRNA reached the most pronounced hazard ratio (HR=2.07; p = 0.05), followed by VEGF-C mRNA (HR=1.59; p = 0.005). HR values above 1.0 indicate that high levels of either CDH5 or VEGF-C mRNAs associated with a higher risk of poor clinical outcome. Distant recurrence incidence was 26% for the CDH5high and 3% for the CDH5low subgroup (Kaplan-Meier analysis). Distant recurrence incidence was 23% for the VEGF-Chigh and 0% for VEGF-Clow subgroup. The independent prognostic value of VEGF-C mRNA was confirmed by LASSO regression. CONCLUSION: Intratumoral VEGF-A levels did not associate with disease outcome in primary, early-stage, breast cancer patients, whilst raised levels of either CDH5 or VEGF-C prognosticated a high risk of distant metastasis.

Development of a dual-flow tissue perfusion device for modeling the gastrointestinal tract-brain axis.

Despite the large number of microfluidic devices that have been described over the past decade for the study of tissues and organs, few have become widely adopted. There are many reasons for this lack of adoption, primarily that devices are constructed for a single purpose or because they are highly complex and require relatively expensive investment in facilities and training. Here, we describe a microphysiological system (MPS) that is simple to use and provides fluid channels above and below cells, or tissue biopsies, maintained on a disposable, poly(methyl methacrylate), carrier held between polycarbonate outer plates. All other fittings are standard Luer sizes for ease of adoption. The carrier can be coated with cells on both sides to generate membrane barriers, and the devices can be established in series to allow medium to flow from one cell layer to another. Furthermore, the carrier containing cells can be easily removed after treatment on the device and the cells can be visualized or recovered for additional off-chip analysis. A 0.4 µm membrane with cell monolayers proved most effective in maintaining separate fluid flows, allowing apical and basal surfaces to be perfused independently. A panel of different cell lines (Caco-2, HT29-MTX-E12, SH-SY5Y, and HUVEC) were successfully maintained in the MPS for up to 7 days, either alone or on devices connected in series. The presence of tight junctions and mucin was expressed as expected by Caco-2 and HT-29-MTX-E12, with Concanavalin A showing uniform staining. Addition of Annexin V and PI showed viability of these cells to be >80% at 7 days. Bacterial extracellular vesicles (BEVs) produced by Bacteroides thetaiotaomicron and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate (DiD) were used as a model component of the human colonic microbiota and were visualized translocating from an apical surface containing Caco-2 cells to differentiated SH-SY5Y neuronal cells cultured on the basal surface of connected devices. The newly described MPS can be easily adapted, by changing the carrier to maintain spheroids, pieces, or slices of biopsy tissue and joined in series to study a variety of cell and tissue processes. The cell layers can be made more complex through the addition of multiple cell types and/or different patterning of extracellular matrix and the ability to culture cells adjacent to one another to allow study of cell:cell transfer, e.g., passive or active drug transfer, virus or bacterial entry or BEV uptake and transfer.

Head and Neck Squamous Cell Carcinoma Biopsies Maintained Ex Vivo on a Perfusion Device Show Gene Changes with Time and Clinically Relevant Doses of Irradiation.

Advancements in 3-Dimensional (3D) culture models for studying disease have increased significantly over the last two decades, but fully understanding how these models represent in vivo still requires further investigation. The current study investigated differences in gene expression between a baseline sample and that maintained on a tissue-on-chip perfusion device for up to 96 h, with and without clinically-relevant doses of irradiation, to allow differentiation of model and treatment effects. Tumour tissue samples from 7 Head and Neck Squamous Cell Carcinomas (HNSCC) patients were sub-divided and either fixed immediately upon excision or maintained in a tissue-on-chip device for 48 and 96 h, with or without 2 Gray (Gy) or 10 Gy irradiation. Gene expression was measured using an nCounter® PanCancer Progression Panel. Differentially expressed genes between pre- and post-ex vivo culture, and control and irradiated samples were identified using nSolver software (version 4.0). The secretome from the tumour-on-chip was analysed for the presence of cytokines using a Proteome Profiler™ platform. Significant numbers of genes both increased (n = 6 and 64) and decreased (n = 18 and 58) in expression in the tissue maintained on-chip for 48 and 96 h, respectively, compared to fresh tissue; however, the irradiation schedule chosen did not induce significant changes in gene expression or cytokine secretion. Although HNSCC tissue maintained ex vivo shows a decrease in a large proportion of altered genes, 25% and 53% (48 and 96 h) do show increased expression, suggesting that the tissue remains functional. Irradiation of tumour tissue-on-chip needs to be conducted for longer time periods for specific gene changes to be observed, but we have shown, for the first time, the feasibility of using this perfusion platform for studying the genomic response of HNSCC tissue biopsies.

Investigating the effects of arginine methylation inhibitors on microdissected brain tumour biopsies maintained in a miniaturised perfusion system.

Arginine methylation is a post-translational modification that consists of the transfer of one or two methyl (CH3) groups to arginine residues in proteins. Several types of arginine methylation occur, namely monomethylation, symmetric dimethylation and asymmetric dimethylation, which are catalysed by different protein arginine methyltransferases (PRMTs). Inhibitors of PRMTs have recently entered clinical trials to target several types of cancer, including gliomas (NCT04089449). People with glioblastoma (GBM), the most aggressive form of brain tumour, are among those with the poorest quality of life and likelihood of survival of anyone diagnosed with cancer. There is currently a lack of (pre)clinical research on the possible application of PRMT inhibitors to target brain tumours. Here, we set out to investigate the effects of clinically-relevant PRMT inhibitors on GBM biopsies. We present a new, low-cost, easy to fabricate perfusion device that can maintain GBM tissue in a viable condition for at least eight days post-surgical resection. The miniaturised perfusion device enables the treatment of GBM tissue with PRMT inhibitors ex vivo, and we observed a two-fold increase in apoptosis in treated samples compared to parallel control experiments. Mechanistically, we show thousands of differentially expressed genes after treatment, and changes in the type of arginine methylation of the RNA binding protein FUS that are consistent with hundreds of differential gene splicing events. This is the first time that cross-talk between different types of arginine methylation has been observed in clinical samples after treatment with PRMT inhibitors.

The Use of Tissue-on-Chip Technology to Focus the Search for Extracellular Vesicle miRNA Biomarkers in Thyroid Disease.

Small extracellular vesicles (sEVs) contain microRNAs (miRNAs) which have potential to act as disease-specific biomarkers. The current study uses an established method to maintain human thyroid tissue ex vivo on a tissue-on-chip device, allowing the collection, isolation and interrogation of the sEVs released directly from thyroid tissue. sEVs were analysed for differences in miRNA levels released from benign thyroid tissue, Graves' disease tissue and papillary thyroid cancer (PTC), using miRNA sequencing and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to identify potential biomarkers of disease. Thyroid biopsies from patients with benign tissue (n = 5), Graves' disease (n = 5) and PTC (n = 5) were perfused with medium containing sEV-depleted serum for 6 days on the tissue-on-chip device. During incubation, the effluents were collected and ultracentrifuged to isolate sEVs; miRNA was extracted and sequenced (miRNASeq). Out of the 15 samples, 14 passed the quality control and miRNASeq analysis detected significantly higher expression of miR-375-3p, miR-7-5p, miR-382-5p and miR-127-3p in the sEVs isolated from Graves' tissue compared to those from benign tissue (false discovery rate; FDR p < 0.05). Similarly, miR-375-3p and miR-7-5p were also detected at a higher level in the Graves' tissue sEVs compared to the PTC tissue sEVs (FDR p < 0.05). No significant differences were observed between miRNA in sEVs from PTC vs. those from benign tissue. These results were supported by Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). The novel findings demonstrate that the tissue-on-chip technology is a robust method for isolating sEVs directly from the tissue of interest, which has permitted the identification of four miRNAs, with which further investigation could be used as biomarkers or therapeutic targets within thyroid disease.

Can granulysin provide prognostic value in primary breast cancer?

BACKGROUND: Granulysin (GNLY) is a cytolytic and proinflammatory molecule which also acts as an immune alarmin. The multifunctional nature of this molecule has made it challenging to define its full potential as a biomarker in breast cancer. AIM: To evaluate the prognostic value of intratumoral GNLY in primary breast cancer patients and its association with established clinicopathological parameters. PATIENTS AND METHODS: The study included 69 node-negative breast cancer patients with known clinicopathological parameters, all of whom had not received any prior hormonal or chemotherapeutic systemic therapy that would interfere with the course of disease. The median follow-up period was 144 months. Steroid hormone receptor status was determined by ligand-binding assay and HER2 status by chromogenic in situ hybridisation (CISH). Intratumoral GNLY mRNA levels were determined by RT-qPCR. Prognostic performance was evaluated by the receiver operating characteristic (ROC), Cox proportional hazards regression and Kaplan-Meier analysis. Classification of patients into GNLYlow and GNLYhigh subgroups was performed by the use of the outcome-oriented cut-off point categorisation approach. RESULTS: There was a significant difference between GNLY values of patients without any recurrences and those with local or distant recurrences (Mann-Whitney test, p = 0.05 and p = 0.02, respectively). None of the tested parameters showed prognostic significance for local and distant recurrences when combined. When distant metastases and local recurrences were separated as events, the best prognostic performance was observed for GNLY as compared with any clinicopathological parameter (AUC=0.24 and p = 0.04 for local events; AUC=0.71 and p = 0.03 for distant events). Local recurrence incidence was 0% for the GNLYhigh subgroup and 19% for the GNLYlow subgroup; however distant recurrence incidence was 24% for the GNLYhigh subgroup but only 3% for the GNLYlow subgroup (Kaplan-Meier analysis). A significant positive correlation was found between intratumoral ER and GNLY levels, and a significant negative correlation between tumour grade and GNLY levels. CONCLUSION: High levels of granulysin prognosticate low risk of local recurrence but a high risk of distant metastasis in primary, untreated, breast cancer patients.

Prevention and removal of membrane and separator biofouling in bioelectrochemical systems: a comprehensive review.

Bioelectrochemical systems (BESs) have made significant progress in recent years in all aspects of their technology. BESs usually work with a membrane or a separator, which is one of their most critical components affecting performance. Quite often, biofilm from either the anolyte or catholyte forms on the membrane, which can negatively affect its performance. In critical cases, the long-term power performance observed for microbial fuel cells (MFCs) has dropped by over 90%. Surface modification and composite material approaches as well as chemical and physical cleaning techniques involving surfactants, acids, hydroxides, and ultrasounds have been successfully implemented to combat biofilm formation. Surface modifications produced up to 6-7 times higher power performance in the long-term, whereas regeneration strategies resulted in up to 100% recovery of original performance. Further studies include tools such as fluid dynamics-based design and plasma cleaning. The biofouling area is still underexplored in the field of bioelectrochemistry and requires systematic improvement. Therefore, this review summarizes the most recent knowledge with the aim of helping the research and engineering community select the best strategy and discuss further perspectives for combating the undesirable biofilm.

The prognostic significance of serum interferon-gamma (IFN-γ) in hormonally dependent breast cancer.

BACKGROUND: Interferon-γ (IFN-γ) is a pleiotropic immunomodulatory cytokine. Because of its contradictory and even dualistic roles in malignancies, its potential as a biomarker remains to be unraveled. AIM: To evaluate the prognostic significance of serum IFN-γ in hormonally treated breast cancer patients. MATERIAL AND METHODS: The study included 72 premenopausal breast cancer patients with known clinicopathological characteristics. All patients received adjuvant hormonal therapy based on hormone receptor-positivity. The median follow-up period was 93 months. IFN-γ serum protein levels were determined by quantitative ELISA. Prognostic performance was evaluated by the receiver operating characteristic (ROC), Cox proportional hazards regression and Kaplan-Meier analyses. Classification of patients into IFN-γlow and IFN-γhigh subgroups was performed by the use of the outcome-oriented cut-off point categorization approach. RESULTS: The best prognostic performance was achieved by IFN-γ (AUC = 0.24 and p = 0.01 for distant events, AUC = 0.29 and p = 0.01 for local and distant events combined). Age and IFN-γ were prognostically significant in instances of all types of outcomes and IFN-γ was the independent prognostic parameter (Cox regression). There was a significant difference between IFN-γ values of patients without any events and those with distant metastases (Mann-Whitney test, p = 0.007). IFN-γ levels correlated significantly with nodal status and tumor stage (Spearman's rank order, r = -0.283 and r = -0.238, respectively). Distant recurrence incidence was 4% for the IFN-γhigh subgroup and 33% for the IFN-γlow subgroup (Kaplan-Meier analysis). CONCLUSIONS: Raised serum IFN-γ levels associate independently with favorable disease outcome in hormonally dependent breast cancer.

Microbial fuel cell scale-up options: Performance evaluation of membrane (c-MFC) and membrane-less (s-MFC) systems under different feeding regimes.

In recent years, bioelectrochemical systems have advanced towards upscaling applications and tested during field trials, primarily for wastewater treatment. Amongst reported trials, two designs of urine-fed microbial fuel cells (MFCs) were tested successfully on a pilot scale as autonomous sanitation systems for decentralised area. These designs, known as ceramic MFCs ( c -MFCs) and self-stratifying MFCs ( s -MFC), have never been calibrated under similar conditions. Here, the most advanced versions of both designs were assembled and tested under similar feeding conditions. The performance and efficiency were evaluated under different hydraulic retention times (HRT), through chemical oxygen demand measures and polarisation experiments. Results show that c -MFCs displayed constant performance independently from the HRT (32.2 ± 3.9 W m-3) whilst displaying high energy conversion efficiency at longer HRT (NER COD  = 2.092 ± 0.119 KWh.Kg COD -1, at 24h HRT). The s -MFC showed a correlation between performance and HRT. The highest performance was reached under short HRT (69.7 ± 0.4 W m-3 at 3h HRT), but the energy conversion efficiency was constant independently from the HRT (0.338 ± 0.029 KWh.Kg COD -1). The c -MFCs and s -MFCs similarly showed the highest volumetric efficiency under long HRT (65h) with NER V of 0.747 ± 0.010 KWh.m-3 and 0.825 ± 0.086 KWh.m-3, respectively. Overall, c -MFCs seems more appropriate for longer HRT and s -MFCs for shorter HRT.

Microbial fuel cell compared to a chemostat.

Microbial Fuel Cells (MFCs) represent a green and sustainable energy conversion system that integrate bacterial biofilms within an electrochemical two-electrode set-up to produce electricity from organic waste. In this review, we focus on a novel exploratory model, regarding "thin" biofilms forming on highly perfusable (non-diffusible) anodes in small-scale, continuous flow MFCs due to the unique properties of the electroactive biofilm. We discuss how this type of MFC can behave as a chemostat in fulfilling common properties including steady state growth and multiple steady states within the limit of biological physicochemical conditions imposed by the external environment. With continuous steady state growth, there is also continuous metabolic rate and continuous electrical power production, which like the chemostat can be controlled. The model suggests that in addition to controlling growth rate and power output by changing the external resistive load, it will be possible instead to change the flow rate/dilution rate.

Microbial fuel cells and their electrified biofilms.

Bioelectrochemical systems (BES) represent a wide range of different biofilm-based bioreactors that includes microbial fuel cells (MFCs), microbial electrolysis cells (MECs) and microbial desalination cells (MDCs). The first described bioelectrical bioreactor is the Microbial Fuel Cell and with the exception of MDCs, it is the only type of BES that actually produces harvestable amounts of electricity, rather than requiring an electrical input to function. For these reasons, this review article, with previously unpublished supporting data, focusses primarily on MFCs. Of relevance is the architecture of these bioreactors, the type of membrane they employ (if any) for separating the chambers along with the size, as well as the geometry and material composition of the electrodes which support biofilms. Finally, the structure, properties and growth rate of the microbial biofilms colonising anodic electrodes, are of critical importance for rendering these devices, functional living 'engines' for a wide range of applications.

Microbial Fuel Cell Based Thermosensor for Robotic Applications.

On the roadmap to building completely autonomous artificial bio-robots, all major aspects of robotic functions, namely, energy generation, processing, sensing, and actuation, need to be self-sustainable and function in the biological realm. Microbial Fuel Cells (MFCs) provide a platform technology for achieving this goal. In a series of experiments, we demonstrate that MFCs can be used as living, autonomous sensors in robotics. In this work, we focus on thermal sensing that is akin to thermoreceptors in mammalian entities. We therefore designed and tested an MFC-based thermosensor system for utilization within artificial bio-robots such as EcoBots. In open-loop sensor characterization, with a controlled load resistance and feed rate, the MFC thermoreceptor was able to detect stimuli of 1 min directed from a distance of 10 cm causing a temperature rise of ∼1°C at the thermoreceptor. The thermoreceptor responded to continuous stimuli with a minimum interval of 384 s. In a practical demonstration, a mobile robot was fitted with two artificial thermosensors, as environmental thermal detectors for thermotactic application, mimicking thermotaxis in biology. In closed-loop applications, continuous thermal stimuli were detected at a minimum time interval of 160 s, without the need for complete thermoreceptor recovery. This enabled the robot to detect thermal stimuli and steer away from a warmer thermal source within the rise of 1°C. We envision that the thermosensor can be used for future applications in robotics, including as a potential sensor mechanism for maintaining thermal homeostasis.

Effect of simple interventions on the performance of a miniature MFC fed with fresh urine.

The aim of the present study is to enhance the performance of a microbial fuel cell (MFC) design by making simple interventions. Specifically, terracotta "t" and mullite "m" ceramics are tested as membranes while carbon veil and carbon cloth are used as electrodes. In the case of "m" cylinders different dimensions are examined (m: ID 30 mm x height 11.5 mm; sm: ID 18 mm x height 18 mm). The units operated continuously with urine as the feedstock. The best performing is the sm type (60-100 µW), followed by the t type (40-80 µW) and the m type (20-40 µW). Polarisation experiments indicated that activated carbon on the anode enhances the power output (t: 423 µW, sm: 288 µW). Similarly, the increase of the surface area and the addition of stainless steel mesh on the cathode improves the power performance for the "sm" and the "t" units. Furthermore, it is shown that the design with the smaller internal diameter, performs better and is more stable through time.

Identification of soluble tissue-derived biomarkers from human thyroid tissue explants maintained on a microfluidic device.

Although a large cohort of potential biomarkers for thyroid cancer aggressiveness have been tested in various formats in recent years, to the best of our knowledge, thyroglobulin and calcitonin remain the only two established biomarkers associated with thyroid cancer management. Our group has recently validated a novel means of maintaining live, human ex vivo thyroid tissue within a tissue-on-chip format. The present pilot study aimed to interrogate the tissue effluent, containing all the soluble markers released by the tissue samples maintained within the devices' tissue chamber, for the presence of markers potentially associated with thyroid cancer aggressiveness. Culture effluent from tissue samples harvested from 19 individual patients who had undergone thyroidectomy for the treatment of suspected thyroid cancer was assessed, first using a proteome profiler™ angiogenesis array kit. Patients were subcategorised as 'aggressive' if they possessed a minimum of N1b level metastases, whilst 'non-aggressive' samples were T3 or lower without evidence of multifocality; and contralateral healthy thyroid tissue was harvested for comparative studies. Levels of Serpin-F1, vascular endothelial growth factor, Thrombospondin-1 and chemokine (C-C motif) ligand were significantly altered and, thus, were further investigated using ELISA to allow for quantitative analysis. The concentration of serpin-F1 was significantly increased in the effluent of aggressive thyroid cancer tissue when compared with levels released by both non-aggressive and benign samples. The present study demonstrated the usability of microfluidic technology for the analysis of the ex vivo tissue secretome in order to identify novel biomarkers.

Electronic faucet powered by low cost ceramic microbial fuel cells treating urine.

Hygienic measures are extremely important to avoid the transmission of contagious viruses and diseases. The use of an electronic faucet increases the hygiene, encourages hand washing, avoids touching the faucet for opening and closing, and it saves water, since the faucet is automatically closed. The microbial fuel cell (MFC) technology has the capability to convert environmental waste into energy. The implementation of low cost ceramic MFCs into electronic interfaces integrated in toilets, would offer a compact powering system as well as an environmentally friendly small-scale treatment plant. In this work, the use of low cost ceramic MFCs to power an L20-E electronic faucet is presented for the first time. A single MFC was capable of powering an electronic faucet with an open/close cycle of 8.5 min, with 200 ml of urine. With a footprint of 360 cm3, the MFC could easily be integrated in a toilet. The possibility to power e-toilet components with MFCs offers a sustainable energy generation system. Other electronic components including an automatic flush, could potentially be powered by MFCs and contribute to the maintenance efficiency and hygiene of the public toilets, leading to a new generation of self-sustained energy recovering e-toilets.

Electrosynthesis, modulation, and self-driven electroseparation in microbial fuel cells.

Microbial electrosynthesis (MES) represents a sustainable platform that converts waste into resources, using microorganisms within an electrochemical cell. Traditionally, MES refers to the oxidation/reduction of a reactant at the electrode surface with externally applied potential bias. However, microbial fuel cells (MFCs) generate electrons that can drive electrochemical reactions at otherwise unbiased electrodes. Electrosynthesis in MFCs is driven by microbial oxidation of organic matter releasing electrons that force the migration of cationic species to the cathode. Here, we explore how electrosynthesis can coexist within electricity-producing MFCs thanks to electro-separation of cations, electroosmotic drag, and oxygen reduction within appropriately designed systems. More importantly, we report on a novel method of in situ modulation for electrosynthesis, through additional "pin" electrodes. Several MFC electrosynthesis modulating methods that adjust the electrode potential of each half-cell through the pin electrodes are presented. The innovative concept of electrosynthesis within the electricity producing MFCs provides a multidisciplinary platform converting waste-to-resources in a self-sustainable way.

Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms.

Procoagulant activity of tissue factor (TF) in response to injury or inflammation is accompanied with cellular signals which determine the fate of cells. However, to prevent excessive signalling, TF is rapidly dissipated through release into microvesicles, and/or endocytosis. To elucidate the mechanism by which TF signalling may become moderated on the surface of cells, the associations of TF, fVII/fVIIa, PAR2 and caveolin-1 on MDA-MB-231, BxPC-3 and 786-O cells were examined and compared to that in cells lacking either fVII/fVIIa or TF. Furthermore, the localisation of labelled-recombinant TF with cholesterol-rich lipid rafts was explored on the surface of primary human blood dermal endothelial cells (HDBEC). Finally, by disrupting the caveolae on the surface of HDBEC, the outcome on TF-mediated signalling was examined. The association between TF and PAR2 was found to be dependent on the presence of fVIIa. Interestingly, the presence of TF was not pre-requisite for the association between fVII/fVIIa and PAR2 but was significantly enhanced by TF, which was also essential for the proliferative signal. Supplementation of HDBEC with exogenous TF resulted in early release of fVII/fVIIa from caveolae, followed by re-sequestration of TF-fVIIa. Addition of labelled-TF resulted in the accumulation within caveolin-1-containing cholesterol-rich regions and was also accompanied with the increased assimilation of cell-surface fVIIa. Disruption of the caveolae/rafts in HDBEC using MßCD enhanced the TF-mediated cellular signalling. Our data supports a hypothesis that cells respond to the exposure to TF by moderating the signalling activities as well as the procoagulant activity of TF, through incorporation into the caveolae/lipid rafts.

De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions.

In this study, the role of de-palmitoylation of tissue factor (TF) in the decryption of its activity was explored. TF-tGFP constructs were prepared by mutagenesis-substitution at Cys245 to prevent or mimic palmitolyation. Additionally, to reduce TF de-palmitoylation, the expression of palmitoyl-protein thioesterases (PPT) was suppressed. Other TF mutants were prepared with altered flexibility, hydrophobicity or length of the transmembrane domain. The outcome of these alterations on fXa-generation, fVIIa binding, Ser253 phosphorylation and TF-microvesicle release were assessed in endothelial cells, and the influence on endothelial and MCF-7 cell proliferation and apoptosis was analysed. Preventing TF palmitoylation (TFSer245-tGFP), increasing the hydrophobicity (TFPhe241-tGFP) or lengthening (TFLongTM-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TFWt-tGFP, but fXa-generation was not further increased following PAR2 activation. Extending the available length of the transmembrane domain enhanced the TF-tGFP release within microvesicles and Ser253 phosphorylation and increased cell proliferation. Moreover, prevention of PKCα-mediated Ser253 phosphorylation with Gö6976 did not preclude fXa-generation. Conversely, reducing the hydrophobicity (TFSer242-tGFP), shortening (TFShortTM-tGFP) or reducing the flexibility (TFVal225-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activation. PPT knock-down or mimicking palmitoylation (TFPhe245-tGFP) reduced fXa-generation without affecting fVIIa binding. This study has for the first time shown that TF procoagulant activity is regulated through de-palmitoylation, which alters the orientation of its transmembrane domain and is independent of TF phosphorylation. However, Ser253 phosphorylation is facilitated by changes in the orientation of the transmembrane domain and can induce TF-cellular signalling that influences cellular proliferation/apoptosis.

Current understanding of nonsurgical interventions for refractory differentiated thyroid cancer: a systematic review.

Thyroid cancer incidence and related mortality is increasing year-on-year, and although treatment for early disease with surgery and radioiodine results in a 98% 5-year survival rate, recurrence and treatment refractory disease is evident in an unacceptable number of patients. Alternative treatment regimens have therefore been sought in the form of tyrosine kinase inhibitors, immunotherapy, vaccines, chimeric antigen receptor T-cell therapy and oncolytic viruses. The current review aims to consolidate knowledge and highlight the latest clinical trials using secondary therapies in thyroid cancer treatment, focusing on both in vitro and in vivo studies, which have investigated therapies other than radioiodine.