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In this first systematic investigation of mechanochemical polyoxometalate (POM) reduction, (TBA) 3 [PMo 12 O 40 ] was reacted with n equiv of lithium metal ( n = 1-24) to generate PMo 12 / n products which were shown to be mixtures of electron-rich PMo 12 Li x species. FTIR analysis revealed the lengthening/weakening of terminal Mo═O bonds with increasing levels of reduction, while EXAFS spectra indicated the onset of Mo-Mo bond formation at n ∼ 8 and a significant structural change at n > 12. Successive Mo VI reductions were monitored by XANES and XPS, and at n = 24, results were consistent with the formation of at least one Mo IV -Mo IV bonded {Mo IV 3 } triad together with Mo V . Upon dissolution, the PMo 12 Li x species present in the solid PMo 12 / n products undergo electron exchange and single-peak 31 P NMR spectra were observed for n = 1-12. For n ≥ 16, changes in solid state and solution 31 P NMR spectra coincided with the emergence of features in the UV-vis spectra associated with Mo V -Mo V and {Mo IV 3 } bonding in an ε-Keggin structure. Bonding between {Li(NCMe)} + and 2-electron-reduced PMo 12 in (TBA) 4 [PMo 12 O 40 {Li(NCMe)}] suggests that super-reduction gives rise to more extensive Li-O bonding that ultimately causes lithium-oxide-promoted TBA cation decomposition and POM degradation, which might explain the appearance of XPS peaks for Mo 2 C at n ≥ 16. This work has revealed some of the complex, unexplored chemistry of super-reduced POMs and establishes a new, solvent-free approach in the search for a better fundamental understanding of the electronic properties and reactivity of electron-rich nanoscale metal oxides.

Chemiresitive sensing allows the affordable and facile detection of small molecules such as H 2 O and CO 2 . Herein, we report a novel class of Earth-abundant post transition metal substituted Keggin polyoxometalates (POMs) for chemiresistive sensing applications, with conductivities up to 0.01 S cm -1 under 100% CO 2 and 65% Relative Humidity (RH).

A new isopolyoxotungstate has been characterised, thirty years since the first spectroscopic evidence of its existence. The heptatungstate [W 7 O 24 H] 5- , containing a {W 5 } lacunary Lindqvist unit fused to a ditungstate fragment, has significant stability and is only the third isopolytungstate structure to be obtained from non-aqueous systems.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non-invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell-derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor-matched pairs of adjacent-normal versus disease-associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease-discriminating mRNA. A set of transcripts including Caveolin-1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression-free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV-associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in-silico model was produced, showcasing the superiority of this multi-modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

The exploration of composition-structure-function relationship in proton-conducting solids remains a challenge in materials chemistry. Polyoxometalate-based compounds have been long considered as candidates for proton conductors; however, their low structural stability and a large decrease in conductivity under reduced relative humidity (RH) have limited their applications. To overcome such limitations, the hybridization of polyoxometalates with proton-conducting polymers has emerged as a promising method. Besides, 4f lanthanide ions possess a high coordination number, which can be utilized to attract water molecules and to build robust frameworks. Herein, a Preyssler-type polyoxometalate functionalized with a 9-coordinate Eu 3+ (Eu[P 5 W 30 O 110 K] 11- ) is newly synthesized and combined with poly(allylamine) with amine moieties as protonation sites. The resulting robust crystalline composite exhibits an ultrahigh proton conductivity >10 -2 S cm -1 at 368 K and 90% RH, which is still >10 -3 S cm -1 at 50% RH, due to the strengthened and extended hydrogen-bonding network.

Aquaporins are required by cells to enable fast adaptation to volume and osmotic changes, as well as microenvironmental metabolic stimuli. Aquaglyceroporins play a crucial role in supplying cancer cells with glycerol for metabolic needs. Here, we show that AQP3 is differentially expressed in cells of a prostate cancer panel. AQP3 is located at the cell membrane and cytoplasm of LNCaP cell while being exclusively expressed in the cytoplasm of Du145 and PC3 cells. LNCaP cells show enhanced hypoxia growth; Du145 and PC3 cells display stress factors, indicating a crucial role for AQP3 at the plasma membrane in adaptation to hypoxia. Hypoxia, both acute and chronic affected AQP3's cellular localization. These outcomes were validated using a machine learning classification approach of the three cell lines and of the six normoxic or hypoxic conditions. Classifiers trained on morphological features derived from cytoskeletal and nuclear labeling alongside corresponding texture features could uniquely identify each individual cell line and the corresponding hypoxia exposure. Cytoskeletal features were 70-90% accurate, while nuclear features allowed for 55-70% accuracy. Cellular texture features (73.9% accuracy) were a stronger predictor of the hypoxic load than the AQP3 distribution (60.3%).

Background: Osteosarcoma is an aggressive and painful bone neoplasm in dogs. Previous studies have reported epidemiological associations suggesting that large body mass, long bone length and the genetics of certain breeds including the Rottweiler are associated with elevated osteosarcoma risk. However, these studies were often limited by selection bias and confounding factors, and have rarely offered insights into breed-associated protection for osteosarcoma. The current study includes 1756 appendicular and axial osteosarcoma cases presenting to VPG Histology (Bristol, UK) compared against a control population of 905,211 dogs without osteosarcoma from primary care electronic patient records in the VetCompass™ dataset., Methods and Study Design: Retrospective, case-control study. Multivariable logistic regression analysis explored associations between demographic risk factors (including breed, chondrodystrophy, age, sex/neuter status, skull-shape, and body mass) and osteosarcoma of all anatomical sites., Results: We identified several breeds with increased and reduced odds of osteosarcoma. At highest risk were the Rottweiler and Great Dane, with > 10 times the odds of osteosarcoma compared with crossbreds, and the Rhodesian Ridgeback, which has not featured in previous lists of at-risk breeds for osteosarcoma, and had an odds ratio of 11.31 (95% confidence interval 7.37-17.35). Breeds at lowest risk of osteosarcoma (protected breeds) included the Bichon Frise, the French Bulldog and the Cavalier King Charles Spaniel, all with odd ratios of less than 0.30 compared with crossbreds. Body mass was strongly associated with osteosarcoma risk; dogs over 40 kg exhibited osteosarcoma odds of 45.44 (95% confidence interval 33.74-61.20) compared with dogs less than 10 kg. Chondrodystrophic breeds had an osteosarcoma odds ratio of 0.13 (95% confidence interval 0.11-0.16) compared with non-chondrodystrophic breeds., Conclusions: This study provides evidence of strong breed-associated osteosarcoma risk and protection, suggesting a genetic basis for osteosarcoma pathogenesis. It highlights that breeds selected for long legs/large body mass are generally overrepresented amongst at-risk breeds, whilst those selected for short leg length/small body mass are generally protected. These findings could inform genetic studies to identify osteosarcoma risk alleles in canines and humans; as well as increasing awareness amongst veterinarians and owners, resulting in improved breeding practices and clinical management of osteosarcoma in dogs.

Amphiphilic polyoxometalate (POM) surfactants were prepared by covalently grafting double hydrophobic tails with chain lengths C12H25, C14H29, C16H33 or C18H37 onto the lacunary Wells-Dawson {P2W17O61} headgroup. The critical micelle concentrations (CMCs) of these novel surfactants in aqueous solutions were determined by conductivity, and micelle formation was studied by small angle neutron scattering (SANS). Surprisingly, the amphiphiles with longer hydrophobic tails tend to form less elongated and more globular micelles in water. The self-assembled amphiphilic polyoxometalates were used as templates in the hydrothermal synthesis of mesoporous TiO2 containing dispersed, immobilised {P2W17O61} units, which showed enhanced activity for the photodegradation of rhodamine B (RhB). The catalyst was recycled eight times with no loss of efficiency, demonstrating the stability of the hybrid structure. The amphiphilic polyoxometalates, therefore have excellent potential for the synthesis of various types of catalytically active porous materials.

Hypothesis: Polyoxometalates (POMs) are metal oxygen clusters with a range of interesting magnetic and catalytic properties. POMs with attached hydrocarbon chains show amphiphilic behaviour so we hypothesised that mixtures of a nonionic surfactant and anionic surfactants with a polyoxometalate cluster as headgroup would form mixed micelles, giving control of the POM density in the micelle, and which would differ in size and shape from micelles formed by the individual surfactants. Due to the high charge and large size of the POM, we suggested that these would be nonideal mixtures due to the complex interactions between the two types of surfactants. The nonideality and the micellar composition may be quantified using regular solution theory. With supplementary information provided by small-angle neutron scattering (SANS), an understanding of this unusual binary surfactant system can be established., Experiments: A systematic study was performed on mixed surfactant systems containing polyoxometalate-headed amphiphiles (K 10 [P 2 W 17 O 61 OSi 2 (C n H 2n+1 ) 2 ], abbreviated as P 2 W 17 -2C n , where n = 12, 14 or 16) and hexaethylene glycol monododecyl ether (C 12 EO 6 ). Critical micelle concentrations (CMCs) of these mixtures were measured and used to calculate the interaction parameters based on regular solution theory, enabling prediction of micellar composition. Predictions were compared to micelle structures obtained from SANS. A phase diagram was also established., Findings: The CMCs of these mixtures suggest unusual unfavourable interactions between the two species, despite formation of mixed micelles. Micellar compositions obtained from SANS concurred with those calculated using the averaged interaction parameters for P 2 W 17 -2C n /C 12 EO 6 (n = 12 and 14). We attribute the unfavourable interactions to a combination of different phenomena: counterion-mediated interactions between P 2 W 17 units and the unfolding of the ethylene oxide headgroups of the nonionic surfactant, yet micelles still form in these systems due to the hydrophobic interactions between surfactant tails., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Correction for 'Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB' by Roman Mikhaylov et al., Lab Chip, 2020, 20, 1807-1814, DOI: 10.1039/C9LC01192G.

Epoxy-tiglianes are a novel class of diterpene esters. The prototype epoxy-tigliane, EBC-46 (tigilanol tiglate), possesses potent anti-cancer properties and is currently in clinical development as a local treatment for human and veterinary cutaneous tumors. EBC-46 rapidly destroys treated tumors and consistently promotes wound re-epithelialization at sites of tumor destruction. However, the mechanisms underlying these keratinocyte wound healing responses are not completely understood. Here, we investigated the effects of EBC-46 and an analogue (EBC-211) at 1.51 nM-151 µM concentrations, on wound healing responses in immortalized human skin keratinocytes (HaCaTs). Both EBC-46 and EBC-211 (1.51 nM-15.1 µM) accelerated G0/G1-S and S-G2/M cell cycle transitions and HaCaT proliferation. EBC-46 (1.51-151 nM) and EBC-211 (1.51 nM-15.1 µM) further induced significant HaCaT migration and scratch wound repopulation. Stimulated migration/wound repopulation responses were even induced by EBC-46 (1.51 nM) and EBC-211 (1.51-151 nM) with proliferation inhibitor, mitomycin C (1 μM), suggesting that epoxy-tiglianes can promote migration and wound repopulation independently of proliferation. Expression profiling analyses showed that epoxy-tiglianes modulated keratin, DNA synthesis/replication, cell cycle/proliferation, motility/migration, differentiation, matrix metalloproteinase (MMP) and cytokine/chemokine gene expression, to facilitate enhanced responses. Although epoxy-tiglianes down-regulated established cytokine and chemokine agonists of keratinocyte proliferation and migration, enhanced HaCaT responses were demonstrated to be mediated via protein kinase C (PKC) phosphorylation and significantly abrogated by pan-PKC inhibitor, bisindolylmaleimide-1 (BIM-1, 1 μM). By identifying how epoxy-tiglianes stimulate keratinocyte healing responses and re-epithelialization in treated skin, our findings support the further development of this class of small molecules as potential therapeutics for other clinical situations associated with impaired re-epithelialization, such as non-healing skin wounds., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Exosomes (Exo)-based therapy holds promise for treatment of lethal pancreatic cancer (PC). Limited understanding of key factors affecting Exo uptake in PC cells restricts better design of Exo-based therapy. This work aims to study the uptake properties of different Exo by PC cells. Exo from pancreatic carcinoma, melanoma and non-cancer cell lines were isolated and characterised for yield, size, morphology and exosomal marker expression. Isolated Exo were fluorescently labelled using a novel in-house developed method based on copper-free click chemistry to enable intracellular tracking and uptake quantification in cells. Important factors influencing Exo uptake were initially predicted by Design of Experiments (DoE) approach to facilitate subsequent actual experimental investigations. Uptake of all Exo types by PC cells (PANC-1) showed time- and dose-dependence as predicted by the DoE model. PANC-1 cell-derived exosomes (PANC-1 Exo) showed significantly higher uptake in PANC-1 cells than that of other Exo types at the longest incubation time and highest Exo dose. In vivo biodistribution studies in subcutaneous tumour-bearing mice similarly showed favoured accumulation of PANC-1 Exo in self-tissue (i.e. PANC-1 tumour mass) over the more vascularised melanoma (B16-F10) tumours, suggesting intrinsic tropism of PC-derived Exo for their parent cells. This study provides a simple, universal and reliable surface modification approach via click chemistry for in vitro and in vivo exosome uptake studies and can serve as a basis for a rationalised design approach for pre-clinical Exo cancer therapies., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Acoustofluidics has been increasingly applied in biology, medicine and chemistry due to its versatility in manipulating fluids, cells and nano-/micro-particles. In this paper, we develop a novel and simple technology to fabricate a surface acoustic wave (SAW)-based acoustofluidic device by clamping electrodes made using a printed circuit board (PCB) with a piezoelectric substrate. The PCB-based SAW (PCB-SAW) device is systematically characterised and benchmarked with a SAW device made using the conventional photolithography process with the same specifications. Microparticle manipulations such as streaming in droplets and patterning in microchannels were demonstrated in the PCB-SAW device. In addition, the PCB-SAW device was applied as an acoustic tweezer to pattern lung cancer cells to form three or four traces inside the microchannel in a controllable manner. Cell viability of ∼97% was achieved after acoustic manipulation using the PCB-SAW device, which proved its ability as a suitable tool for acoustophoretic applications.

This article was originally published under standard licence, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the paper have been modified accordingly.

Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis.

To find new anti-cancer drug therapies, we wanted to exploit homeostatic vulnerabilities within Tuberous Sclerosis Complex 2 (TSC2)-deficient cells with mechanistic target of rapamycin complex 1 (mTORC1) hyperactivity. We show that nelfinavir and mefloquine synergize to selectively evoke a cytotoxic response in TSC2-deficient cell lines with mTORC1 hyperactivity. We optimize the concentrations of nelfinavir and mefloquine to a clinically viable range that kill cells that lack TSC2, while wild-type cells tolerate treatment. This new clinically viable drug combination causes a significant level of cell death in TSC2-deficient tumor spheroids. Furthermore, no cell recovery was apparent after drug withdrawal, revealing potent cytotoxicity. Transcriptional profiling by RNA sequencing of drug treated TSC2-deficient cells compared to wild-type cells suggested the cytotoxic mechanism of action, involving initial ER stress and an imbalance in energy homeostatic pathways. Further characterization revealed that supplementation with methyl pyruvate alleviated energy stress and reduced the cytotoxic effect, implicating energy deprivation as the trigger of cell death. This work underpins a critical vulnerability with cancer cells with aberrant signaling through the TSC2-mTORC1 pathway that lack flexibility in homeostatic pathways, which could be exploited with combined nelfinavir and mefloquine treatment.

The nuclear-targeting chemical probe, for the detection and quantification of DNA within cells, has been a mainstay of cytometry-from the colorimetric Feulgen stain to smart fluorescent agents with tuned functionality. The level of nuclear structure and function at which the probe aims to readout, or indeed at which a DNA-targeted drug acts, is shadowed by a wide range of detection modalities and analytical methods. These methods are invariably limited in terms of the resolution attainable versus the volume occupied by targeted chromatin structures. The scalar challenge arises from the need to understand the extent and different levels of compaction of genomic DNA and how such structures can be re-modeled, reported, or even perturbed by both probes and drugs. Nuclear cytometry can report on the complex levels of chromatin order, disorder, disassembly, and even active disruption by probes and drugs. Nuclear probes can report defining features of clinical and therapeutic interest as in NETosis and other cell death processes. New cytometric approaches continue to bridge the scalar challenges of analyzing chromatin organization. Advances in super-resolution microscopy address the resolution and depth of analysis issues in cellular systems. Typical of recent insights into chromatin organization enabled by exploiting a DNA interacting probe is ChromEM tomography (ChromEMT). ChromEMT uses the unique properties of the anthraquinone-based cytometric dye DRAQ5™ to reveal that local and global 3D chromatin structures effect differences in compaction. The focus of this review is nuclear and chromatin cytometry, with linked reference to DNA targeting probes and drugs as exemplified by the anthracenediones., (© 2018 International Society for Advancement of Cytometry.)

Structural features from the anticancer prodrug nemorubicin (MMDX) and the DNA-binding molecule DRAQ5™ were used to prepare anthraquinone-based compounds, which were assessed for their potential to interrogate cytochrome P450 (CYP) functional activity and localisation. 1,4-disubstituted anthraquinone 8 was shown to be 5-fold more potent in EJ138 bladder cancer cells after CYP1A2 bioactivation. In contrast, 1,5-bis((2-morpholinoethyl)amino) substituted anthraquinone 10 was not CYP-bioactivated but was shown to be fluorescent and subsequently photo-activated by a light pulse (at a bandwidth 532-587 nm), resulting in punctuated foci accumulation in the cytoplasm. It also showed low toxicity in human osteosarcoma cells. These combined properties provide an interesting prospective approach for opto-tagging single or a sub-population of cells and seeking their location without the need for continuous monitoring., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Incorporation of {MX} n+ groups into polyoxometalates (POMs) provides the means not only to introduce reactivity and functionality but also to tune the electronic properties of the oxide framework by varying M, X and n. In order to elucidate the factors responsible for differences in reactivity between {TiW 5 } and {SnW 5 } Lindqvist-type hexametalates, a series of alkoxido- and aryloxido-tin substituted POMs (nBu 4 N) 3 [(RO)SnW 5 O 18 ] (R=Me, Et, iPr and tBu) and (nBu 4 N) 3 [(ArO)SnW 5 O 18 ] (Ar=C 6 H 5 , 4-MeC 6 H 5 , 4-tBuC 6 H 5 , 4-HOC 6 H 4 , 3-HOC 6 H 4 and 2-CHOC 6 H 4 ) has been structurally characterised and studied by multinuclear NMR ( 1 H, 13 C, 17 O, 119 Sn and 183 W) and FTIR spectroscopy. Spectroscopic and structural parameters were compared with those of titanium-substituted homologues and, when coupled with theoretical studies, indicated that Sn-OR and Sn-OAr bonds are ionic with little π-contribution, whereas Ti-OR and Ti-OAr bonds are more covalent with π-bonding that is more prevalent for Ti-OR than Ti-OAr. This experimental and theoretical analysis of bonding in a homologous series of reactive POMs is the most extensive and detailed to date, and reveals factors which account for significant differences in reactivity between tin and titanium congeners., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: OCT1002 is a unidirectional hypoxia-activated prodrug (uHAP) OCT1002 that can target hypoxic tumor cells. Hypoxia is a common feature in prostate tumors and is known to drive disease progression and metastasis. It is, therefore, a rational therapeutic strategy to directly target hypoxic tumor cells in an attempt to improve treatment for this disease. Here we tested OCT1002 alone and in combination with standard-of-care agents in hypoxic models of castrate-resistant prostate cancer (CRPC)., Methods: The effect of OCT1002 on tumor growth and vasculature was measured using murine PC3 xenograft and dorsal skin fold (DSF) window chamber models. The effects of abiraterone, docetaxel, and cabazitaxel, both singly and in combination with OCT1002, were also compared., Results: The hypoxia-targeting ability of OCT1002 effectively controls PC3 tumor growth. The effect was evident for at least 42 days after exposure to a single dose (30 mg/kg) and was comparable to, or better than, drugs currently used in the clinic. In DSF experiments OCT1002 caused vascular collapse in the PC3 tumors and inhibited the revascularization seen in controls. In this model OCT1002 also enhanced the anti-tumor effects of abiraterone, cabazitaxel, and docetaxel; an effect which was accompanied by a more prolonged reduction in tumor vasculature density., Conclusions: These studies provide the first evidence that OCT1002 can be an effective agent in treating hypoxic, castrate-resistant prostate tumors, either singly or in combination with established chemotherapeutics for prostate cancer., (© 2017 Wiley Periodicals, Inc.)

Uncontrolled cell growth in Tuberous Sclerosis Complex occurs due to inappropriate activation of mechanistic (mammalian) target of rapamycin complex 1 (mTORC1). The current therapy, rapamycin, produced promising clinical trial results, but patient tumours regrow if treatment is discontinued, revealing rapamycin has cytostatic properties rather than a cytotoxic effect. Taking advantage of the enhanced levels of endoplasmic reticulum (ER) stress present in TSC2-null cells, we investigated drug combinations producing a cytotoxic response. We found a nelfinavir and salinomycin combination specifically killed TSC2-deficient, mTORC1 hyperactive cells. Cytotoxicity was rescued by reducing protein synthesis, either through mTORC1 inhibition or cycloheximide treatment. This indicates that the drug combination targets the cells by tipping the protein homeostasis balance of the already metabolically stressed TSC2-deficient cells in favour of cell death. Furthermore, this drug combination also inhibited tumour formation in TSC2-deficient cell models and caused tumour spheroid death in 3D culture. Importantly, the 3D assay could differentiate the cytostatic agent, rapamycin, from the cytotoxic nelfinavir/salinomycin combination. Sporadic cancer cell lines with hyperactive mTORC1 signalling were also susceptible to this nelfinavir/salinomycin drug combination. This work indicates that the protein homeostasis pathway is an attractive therapeutic target in both Tuberous Sclerosis Complex and mTORC1-driven sporadic cancers.

Purpose: To understand the role of hypoxia in prostate tumor progression and to evaluate the ability of the novel unidirectional hypoxia-activated prodrug OCT1002 to enhance the antitumor effect of bicalutamide. Experimental Design: The effect of OCT1002 on prostate cancer cells (LNCaP, 22Rv1, and PC3) was measured in normoxia and hypoxia in vitro In vivo , tumor growth and lung metastases were measured in mice treated with bicalutamide, OCT1002, or a combination. Dorsal skin fold chambers were used to image tumor vasculature in vivo Longitudinal gene expression changes in tumors were analyzed using PCR. Results: Reduction of OCT1002 to its active form (OCT1001) decreased prostate cancer cell viability. In LNCaP-luc spheroids, OCT1002 caused increased apoptosis and decreased clonogenicity. In vivo , treatment with OCT1002 alone, or with bicalutamide, showed significantly greater tumor growth control and reduced lung metastases compared with controls. Reestablishment of the tumor microvasculature following bicalutamide-induced vascular collapse is inhibited by OCT1002. Significantly, the upregulation of RUNX2 and its targets caused by bicalutamide alone was blocked by OCT1002. Conclusions: OCT1002 selectively targets hypoxic tumor cells and enhances the antitumor efficacy of bicalutamide. Furthermore, bicalutamide caused changes in gene expression, which indicated progression to a more malignant genotype; OCT1002 blocked these effects, emphasizing that more attention should be attached to understanding genetic changes that may occur during treatment. Early targeting of hypoxic cells with OCT1002 can provide a means of inhibiting prostate tumor growth and malignant progression. This is of importance for the design and refinement of existing androgen-deprivation regimens in the clinic. Clin Cancer Res; 23(7); 1797-808. ©2016 AACR ., (©2016 American Association for Cancer Research.)

This chapter provides a method for quantitative single cell analysis to track the transition of single cells from G2, indicated by high cyclin B1 levels, to G1 polyploidy phase (G1(p)), indicated by low cyclin B1 levels, in a 4n population. The cell tracking methodology described provides a fluorescence fingerprint suitable for deriving G2/M or G2/G1p transitions. Notably, during late G2 the absolute cyclin B1-eGFP reporter levels obtained were high and the switch-off point identifiable, with destruction rates of a similar order across all cell cycle routing avenues. The three principle parameters extracted were defined as (1) G2-to-G1(p) transition duration (tGFP(off)); (2) rate of sensor destruction (kGFP(off)), and (3) peak sensor expression (GFP(peak)).

Harnessing mesenchymal stem cells for tissue repair underpins regenerative medicine. However, how the 3D tissue matrix maintains such cells in a quiescent state whilst at the same time primed to respond to tissue damage remains relatively unknown. Developing more physiologically relevant 3D models would allow us to better understand the matrix drivers and influence on cell-lineage differentiation in situ. In this study, we have developed an ex vivo organotypic rat mandible slice model; a technically defined platform for the culture and characterization of dental pulp progenitor cells expressing GFP driven by the β-actin promoter (cGFP DPPCs). Using confocal microscopy we have characterized how the native environment influences the progenitor cells transplanted into the dental pulp. Injected cGFP-DPPCs were highly viable and furthermore differentially proliferated in unique regions of the mandible slice; in the dentine region, cGFP-DPPCs showed a columnar morphology indicative of expansion and lineage differentiation. Hence, we demonstrated the systematic capacity for establishing a dental pulp cell-micro-community, phenotypically modified in the tooth (the "biology"); and at the same time addressed technical challenges enabling the mandible slice to be accessible on platforms for high-content imaging (the biology in a "multiplex" format)., (Published by Wiley Periodicals, Inc. on behalf of ISAC.)

Inappropriate activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is common in cancer and has many cellular consequences including elevated endoplasmic reticulum (ER) stress. Cells employ autophagy as a critical compensatory survival mechanism during ER stress. This study utilised drug-induced ER stress through nelfinavir in order to examine ER stress tolerance in cell lines with hyper-active mTORC1 signalling. Our initial findings in wild type cells showed nelfinavir inhibited mTORC1 signalling and upregulated autophagy, as determined by decreased rpS6 and S6K1 phosphorylation, and SQTSM1 protein expression, respectively. Contrastingly, cells with hyper-active mTORC1 displayed basally elevated levels of ER stress which was greatly exaggerated following nelfinavir treatment, seen through increased CHOP mRNA and XBP1 splicing. To further enhance the effects of nelfinavir, we introduced chloroquine as an autophagy inhibitor. Combination of nelfinavir and chloroquine significantly increased ER stress and caused selective cell death in multiple cell line models with hyper-active mTORC1, whilst control cells with normalised mTORC1 signalling tolerated treatment. By comparing chloroquine to other autophagy inhibitors, we uncovered that selective toxicity invoked by chloroquine was independent of autophagy inhibition yet entrapment of chloroquine to acidified lysosomal/endosomal compartments was necessary for cytotoxicity. Our research demonstrates that combination of nelfinavir and chloroquine has therapeutic potential for treatment of mTORC1-driven tumours., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We present a machine learning based approach to automatically detect and segment cells in phase contrast images. The proposed method consists of a multi-stage classification scheme based on random forest (RF) classifier. Both low level and mid level image features are used to determine meaningful cell regions. Pixel-wise RF classification is first carried out to categorize pixels into 4 classes (dark cell, bright cell, halo artifact, and background) and generate a probability map for cell regions. K-means clustering is then applied on the probability map to group similar pixels into candidate cell regions. Finally, cell validation is performed by another RF to verify the candidate cell regions. The proposed method has been tested on U2-OS human osteosarcoma phase contrast images. The experimental results show better performance of the proposed method with precision 92.96% and recall 96.63% compared to a state-of-the-art segmentation technique.

Acidification of an aqueous solution of K8SiW11O39 and K2Pt(OH)6 to pH 4 followed by addition of excess tetramethylammonium (TMA) chloride yielded a solid mixture of TMA salts of H2SiPtW11O40(4-) (1) and SiW12O40(4-) (2). The former was separated from the latter by extraction into an aqueous solution and converted into tetra-n-butylammonium (TBA) and potassium salts TBA-1 and K-1. The α-H2SiPtW11O40(4-) was identified as a monosubstituted Keggin anion using elemental analysis, IR spectroscopy, X-ray crystallography, electrospray ionization mass spectrometry, (195)Pt NMR spectroscopy, (183)W NMR spectroscopy, and (183)W-(183)W 2D INADEQUATE NMR spectroscopy. Both TBA-1 and K-1 readily cocrystallized with their unsubstituted Keggin anion salts, TBA-2 and K-2, respectively, providing an explanation for the historical difficulty of isolating certain platinum-substituted heteropolyanions in pure form.

Motivation: Experimental reproducibility is fundamental to the progress of science. Irreproducible research decreases the efficiency of basic biological research and drug discovery and impedes experimental data reuse. A major contributing factor to irreproducibility is difficulty in interpreting complex experimental methodologies and designs from written text and in assessing variations among different experiments. Current bioinformatics initiatives either are focused on computational research reproducibility (i.e. data analysis) or laboratory information management systems. Here, we present a software tool, ProtocolNavigator, which addresses the largely overlooked challenges of interpretation and assessment. It provides a biologist-friendly open-source emulation-based tool for designing, documenting and reproducing biological experiments., Availability and Implementation: ProtocolNavigator was implemented in Python 2.7, using the wx module to build the graphical user interface. It is a platform-independent software and freely available from http://protocolnavigator.org/index.html under the GPL v2 license., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Dynamic physical interactions between proteins underpin all key cellular processes and are a highly attractive area for the development of research tools and medicines. Protein-protein interactions frequently involve α-helical structures, but peptides matching the sequences of these structures usually do not fold correctly in isolation. Therefore, much research has focused on the creation of small peptides that adopt stable α-helical structures even in the absence of their intended protein targets. We show that short peptides alkylated with azobenzene crosslinkers can be used to photo-stimulate mitochondrial membrane depolarization and cytochrome c release in permeabilised cells, the initial events of the intrinsic apoptosis pathway.

The new tungstatoiodate, α-[H5W18O59(IO3)](6-), containing I(V)O3(-) within a {W18O54} metal oxide framework has been prepared and shown by X-ray crystallography and mass spectrometry to be derived from the fully oxidised [H3W18O56(IO6)](6-) by two-electron reduction accompanied by a redox-triggered structural rearrangement where three I-O covalent bonds are broken.

Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated post-translational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (K i = 10 µM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers.

Discerning the extent of biologically relevant heterogeneity presents unique challenges to both microscopy and flow cytometry. Micro-environmental influences and stochastic changes in cellular behaviour can act to mask the origins of both progression and therapeutic resistance in tumour cell systems. In part the dimensionality of different and frequently metastable states can be assessed by multi-parameter flow cytometry with unparalleled statistical robustness. Complementary application of imaging can provide valuable insights into the complex temporal changes that can occur in cell micro-communities either spontaneously or in response to selection pressure. With an extensive range of methodologies for the labelling of cells there are multiple options for tracking cells, defining fate and the re-construction of provenance and behavioural history. The challenge is highlighted by attempts to identify the critical glycosylation events modifying the function of cell surface proteins. Central to a cytometric approach is the availability of methods that reveal cell health and are compatible with the detection of cell surface changes within dynamic micro-communities. The review briefly addresses the options for sensing cell health and the co-application of an antibody mimetic for detection of cell surface glycoconjugate expression accessible for both imaging and flow cytometry., (© 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.)

Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell-surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence (<30 population doublings) in SCLC cell lines resolved populations with increased (SHP-77 and COR-L279) or negligible (NCI-H69) polysialylation compared with nonadherent parent populations. Adherent forms retained NCAM expression as confirmed by immunofluorescence and immunoblotting. Initial transition to adherence and loss of polysialylation in NCI-H69 was linked to a reduced proliferation rate with no increase in cell death. This reduced proliferation rate was reiterated in vivo as determined by the growth of noninvasive subcutaneous xenografts in mice. Continued selection for enhanced substrate adherence in NCI-H69 (>150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell-cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies., (Copyright © 2013 International Society for Advancement of Cytometry.)

Time-lapse microscopy can be described as the repeated collection of an image (in n-dimensions; x, y, z, λ) or field of view from a microscope at discrete time intervals. The duration of the time interval defines the temporal resolution, which in turn characterizes the type of event detected. This unit describes the implementation of time-lapse microscopy to link initial cell cycle position during acute exposures to anti-cancer agents with anti-proliferative consequences for individual cells. The approach incorporates fundamental concepts arising from the ability to capture simple video sequences of cells from which it is possible to extract kinetic descriptors that reflect the interplay of mitosis and cell death in the growth of an unsynchronized tumor population. Utilizing a multi-well format enables the user to screen different drug derivatives, multiple dose ranges, or cell cultures with unique genetic backgrounds. The objective of this unit is to present the basic methodology for capturing time-lapse sequences and touch upon subsequent mining of the data for deriving event curves and possible cell lineage maps.

We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Dual excitation 488 nm/multiline UV (351-364 nm) flow cytometry was used to confirm ABCG2-specific inhibition of dye efflux using Fumitremorgin C. Transporter gene expression, assayed by qRT-PCR, confirmed higher expression of ABCG2 versus ABCB1, reiterated in a cloned subline. Coexpression of aldehyde dehydrogenase genes ranked as aldehyde dehydrogenase class 1A1 (ALDH1A1) > ALDH3A1 > ALDH3, relative expression of all genes was again reiterated in a cloned subline. Permeabilized cells were used to create red:violet (660:405 nm Em wavelengths) ratiometric references for mapping temporal changes in Hoechst 33342-DNA fluorescence in live cells. A live cell "kinetic SP gate" tracked progressive dye loading of the whole population and coapplication of the far red (>695 nm wavelength) fluorescing dye DRAQ7 enabled viable cell gating. Kinetic gating revealed a continuum for dye accumulation suggesting that SP enumeration is critically dependent upon the nonlinear relationship of the spectral shift with progressive dye-DNA binding and thus requires accurate definition. To this end, permeabilized cell reference samples permit reproducible instrument setup, guide gate boundaries for SP and compromised cells, and offer a simple means of comparing SP enumeration across laboratory sites/platforms. Our approach reports the dynamic range for the spectral shift, revealing noninformative staining conditions and explaining a source of variability for SP enumeration. We suggest that live cell kinetic sorting of all cells with the same dye:DNA load but with differences in efflux capacity can be used to explore drug resistance capability without prejudice. The SP phenotype should be regarded as a kinetic parameter and not a fixed characteristic--critical for functional assay design and the interpretation of heterogeneity., (Copyright © 2012 International Society for Advancement of Cytometry.)

Polysialic acid (polySia) is a carbohydrate polymer critical for neuronal cell migration and axon pathfinding in embryonic development. Besides brain regions requiring persistent neuronal plasticity, polySia is essentially absent from the adult body. However, polySia is aberrantly re-expressed on many tumours, where it decorates the surface of NCAM (neuronal cell adhesion molecule) and modulates cell adhesion, migration and invasion. PolySia-NCAM expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in many cancers, including lung cancer, neuroblastoma and gliomas. The synthesis of polySia is mediated by two polysialyltransferases (polySTs), ST8SiaIV (PST) and particularly ST8SiaII (STX) in cancer cells. The demonstration that polyST knock-down negates events associated with tumour cell dissemination indicates that PST and STX are validated targets. Selective inhibition of polySTs therefore presents a therapeutic opportunity to inhibit tumour invasion and metastasis.

Controlled reductive assembly of capped Keggin anions [PMo(12)O(40)(ML(m))(n)](3-) has been achieved by reduction of [PMo(12)O(40)](3-) with sodium-mercury amalgam in the presence of metal halides, as exemplified by the rational syntheses of mono-capped [PMo(12)O(40){Co(MeCN)(2)}](3-) and bi-capped [PMo(12)O(40)(VO)(2)](3-) and [PMo(12)O(40)Sb(2)](3-)., (This journal is © The Royal Society of Chemistry 2012)

The tetra-n-butylammonium (TBA) salts of [((i)PrO)TiMo(5)O(18)](3-) 1 and [((i)BuO)TiMo(5)O(18)](3-) 2 were prepared by hydrolysis of mixtures of (TBA)(2)[Mo(2)O(7)], (TBA)(4)α-[Mo(8)O(26)] and Ti(OR)(4) (R = (i)Pr or (i)Bu) in acetonitrile. Treatment of (TBA)(3)1 with alcohols ROH afforded primary and tertiary alkoxide derivatives [(RO)TiMo(5)O(18)](3-) (R = Me 3, (t)Bu 4), whilst aryloxides [(ArO)TiMo(5)O(18)](3-) were prepared by reacting 1 with phenols ArOH (Ar = C(6)H(4)Me-4 5, and C(6)H(4)CHO-2 6). Oxo-bridged [(μ-O)(TiMo(5)O(18))(2)](6-)7 rather than the hydroxo derivative [(HO)TiMo(5)O(18)](3-) was obtained upon hydrolysis of 1. X-Ray crystal structures of TBA salts of anions 3-7 show that titanium is six-coordinate in all cases, although titanium sites are disordered over two trans positions in 3. Mo-O bond length alternation is observed in the Mo(4)O(4) planes of 4 and 7 and in one of the two independent anions in the structure of 3. In solution, (17)O NMR spectra are consistent with the higher anionic charge compared to [Mo(6)O(19)](2-) and reveal an order of basicity for the anions [LM'Mo(5)O(18)](3-) associated with the ability of {LM'}(3+) to donate/withdraw electron density from {Mo(5)O(18)}(6-). Protonolysis reactions of 1 and 3 were slower than for tungstate analogues and the possibility of initial protonation at TiOM (M = Mo) rather than TiOR (M = W) in a proton-assisted S(N)1 mechanism for ligand exchange in [(RO)TiM(5)O(18)](3-) is discussed.

An automated technique for the identification, tracking and analysis of biological cells is presented. It is based on the use of nanoparticles, enclosed within intra-cellular vesicles, to produce clusters of discrete, point-like fluorescent, light sources within the cells. Computational analysis of these light ensembles in successive time frames of a movie sequence, using k-means clustering and particle tracking algorithms, provides robust and automated discrimination of live cells and their motion and a quantitative measure of their proliferation. This approach is a cytometric version of the moving light display technique which is widely used for analyzing the biological motion of humans and animals. We use the endocytosis of CdTe/ZnS, core-shell quantum dots to produce the light displays within an A549, epithelial, lung cancer cell line, using time-lapse imaging with frame acquisition every 5 minutes over a 40 hour time period. The nanoparticle moving light displays provide simultaneous collection of cell motility data, resolution of mitotic traversal dynamics and identification of familial relationships allowing construction of multi-parameter lineage trees.

A compartmental model for the in vitro uptake kinetics of the anti-cancer agent topotecan (TPT) has been extended from a previously published model. The extended model describes the drug activity and delivery of the pharmacologically active form to the DNA target as well as the catalysis of the aldehyde dehydrogenase (ALDH) enzyme and the elimination of drug from the cytoplasm via the efflux pump. Verification of the proposed model is achieved using scanning-laser microscopy data from live human breast cancer cells. Before estimating the unknown model parameters from the experimental in vitro data it is essential to determine parameter uniqueness (or otherwise) from this imposed output structure. This is formally performed as a structural identifiability analysis, which demonstrates that all of the unknown model parameters are uniquely determined by the output structure corresponding to the experiment., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In vivo synthesis of peptides by bacterial expression has developed into a reliable alternative to solid-phase peptide synthesis. A significant drawback of in vivo methods is the difficulty with which gene products can be modified post-translationally. Here, we present a method for the facile modification of peptides generated in bacterial hosts after cyanogen bromide cleavage at C-terminal methionines. Reaction of the resulting homoserine lactones with propargylamine allows efficient and selective modification with a wide variety of chemicals such as fluorescent dyes, biotin derivatives, polyprenyls, lipids, polysaccharides, or peptides. Attachment of the cell penetrating peptide octa-arginine (R(8)) to peptides derived from the proapoptotic tumor suppressor Bak BH3 led to efficient cellular uptake and subsequent cytochrome c release from mitochondria, culminating in induction of apoptosis similar to that observed with peptides linked to R(8) via the peptide backbone. These results highlight the significant potential for use of such tools in live cells.

The cell cycle, with its highly conserved features, is a fundamental driver for the temporal control of cell proliferation-while abnormal control and modulation of the cell cycle are characteristic of tumor cells. The principal aim in cancer biology is to seek an understanding of the origin and nature of innate and acquired heterogeneity at the cellular level, driven principally by temporal and functional asynchrony. A major bottleneck when mathematically modeling these biological systems is the lack of interlinked structured experimental data. This often results in the in silico models failing to translate the specific hypothesis into parameterized terms that enable robust validation and hence would produce suitable prediction tools rather than just simulation tools. The focus has been on linking data originating from different cytometric platforms and cell-based event analysis to inform and constrain the input parameters of a compartmental cell cycle model, hence partly measuring and deconvolving cell cycle heterogeneity within a tumor population. Our work has addressed the concept that the interoperability of cytometric data, derived from different cytometry platforms, can complement as well as enhance cellular parameters space, thus providing a more broader and in-depth view of the cellular systems. The initial aim was to enable the cell cycle model to deliver an improved integrated simulation of the well-defined and constrained biological system. From a modeling perspective, such a cross platform approach has provided a paradigm shift from conventional cross-validation approaches, and from a bioinformatics perspective, novel computational methodology has been introduced for integrating and mapping continuous data with cross-sectional data. This establishes the foundation for developing predictive models and in silico tracking and prediction of tumor progression, (Copyright © 2011 International Society for Advancement of Cytometry.)

Background: The inheritance of cellular material between parent and daughter cells during mitosis is highly influential in defining the properties of the cell and therefore the population lineage. This is of particular relevance when studying cell population evolution to assess the impact of a disease or the perturbation due to a drug treatment. The usual technique to investigate inheritance is to use time-lapse microscopy with an appropriate biological marker, however, this is time consuming and the number of inheritance events captured are too low to be statistically meaningful., Results: Here we demonstrate the use of a high throughput fluorescence measurement technique e.g. flow cytometry, to measure the fluorescence from quantum dot markers which can be used to target particular cellular sites. By relating, the fluorescence intensity measured between two time intervals to a transfer function we are able to deconvolve the inheritance of cellular material during mitosis. To demonstrate our methodology we use CdTe/ZnS quantum dots to measure the ratio of endosomes inherited by the two daughter cells during mitosis in the U2-OS, human osteoscarcoma cell line. The ratio determined is in excellent agreement with results obtained previously using a more complex and computational intensive bespoke stochastic model., Conclusions: The use of a transfer function approach allows us to utilise high throughput measurement of large cell populations to derive statistically relevant measurements of the inheritance of cellular material. This approach can be used to measure the inheritance of organelles, proteins etc. and also particles introduced to cells for drug delivery.

The monitoring of cells labeled with quantum dot endosome-targeted markers in a highly proliferative population provides a quantitative approach to determine the redistribution of quantum dot signal as cells divide over generations. We demonstrate that the use of time-series flow cytometry in conjunction with a stochastic numerical simulation to provide a means to describe the proliferative features and quantum dot inheritance over multiple generations of a human tumor population. However, the core challenge for long-term tracking where the original quantum dot fluorescence signal over time becomes redistributed across a greater cell number requires accountability of background fluorescence in the simulation. By including an autofluorescence component, we are able to continue even when this signal predominates (i.e., >80% of the total signal) and obtain valid readouts of the proliferative system. We determine the robustness of the technique by tracking a human osteosarcoma cell population over 8 days and discuss the accuracy and certainty of the model parameters obtained. This systems biology approach provides insight into both cell heterogeneity and division dynamics within the population and furthermore informs on the lineage history of its members., (Copyright © 2010 International Society for Advancement of Cytometry.)

Connexins (Cxs) and gap junction (GJ)-mediated communication have been linked with the regulation of cell cycle traverse. However, it is not clear whether Cx expression or GJ channel function are the key mediators in this process or at what stage this regulation may occur. We therefore tested the hypothesis that enhanced Cx expression could alter the rate of cell cycle traverse independently of GJ channel function. Sodium butyrate (NaBu) or anti-arrhythmic peptide (AAP10) were used to enhance Cx expression in HeLa cells stably expressing Cx43 (HeLa-43) and primary cultures of human fibroblasts (HFF) that predominantly express Cx43. To reduce GJ-mediated communication, 18-alpha-glycyrrhetinic acid (GA) was used. In HeLa-43 and HFF cells, NaBu and AAP10 enhanced Cx43 expression and increased channel function, while GA reduced GJ-mediated communication but did not significantly alter Cx43 expression levels. Timelapse microscopy and flow cytometry of HeLa-WT (wild-type, Cx deficient) and HeLa-43 cells dissected cell cycle traverse and enabled measurements of intra-mitotic time and determined levels of G1 arrest. Enhanced Cx43 expression increased mitotic durations corresponding with a G1 delay in cell cycle, which was linked to an increase in expression of the cell cycle inhibitor p21(waf1/cip1) in both HeLa-43 and HFF cells. Reductions in Cx43 channel function did not abrogate these responses, indicating that GJ channel function was not a critical factor in reducing cell proliferation in either cell type. We conclude that enhanced Cx43 expression and not GJ-mediated communication, is involved in regulating cell cycle traverse., ((c) 2010 Wiley-Liss, Inc.)

We present a new approach to the handling and interrogating of large flow cytometry data where cell status and function can be described, at the population level, by global descriptors such as distribution mean or co-efficient of variation experimental data. Here we link the "real" data to initialise a computer simulation of the cell cycle that mimics the evolution of individual cells within a larger population and simulates the associated changes in fluorescence intensity of functional reporters. The model is based on stochastic formulations of cell cycle progression and cell division and uses evolutionary algorithms, allied to further experimental data sets, to optimise the system variables. At the population level, the in-silico cells provide the same statistical distributions of fluorescence as their real counterparts; in addition the model maintains information at the single cell level. The cell model is demonstrated in the analysis of cell cycle perturbation in human osteosarcoma tumour cells, using the topoisomerase II inhibitor, ICRF-193. The simulation gives a continuous temporal description of the pharmacodynamics between discrete experimental analysis points with a 24 hour interval; providing quantitative assessment of inter-mitotic time variation, drug interaction time constants and sub-population fractions within normal and polyploid cell cycles. Repeated simulations indicate a model accuracy of +/-5%. The development of a simulated cell model, initialized and calibrated by reference to experimental data, provides an analysis tool in which biological knowledge can be obtained directly via interrogation of the in-silico cell population. It is envisaged that this approach to the study of cell biology by simulating a virtual cell population pertinent to the data available can be applied to "generic" cell-based outputs including experimental data from imaging platforms.

Single cell encoding with quantum dots as live cell optical tracers for deriving proliferation parameters has been developed using modelling to investigate cell cycle and proliferative outputs of human osteosarcoma cells undergoing mitotic bypass and endocycle routing. A computer-based simulation of the evolving cell population provides information on the dilution and segregation of nanoparticle dose cell by cell division and allows quantitative assessment of patterns of division, at both single cell and including whole population level cell cycle routing, with no a-priori knowledge of the population proliferation potential. The output therefore provides a unique mitotic distribution function that represents a convolution of cell cycle kinetics (cell division) and the partitioning coefficient for the labelled cell compartment (daughter-daughter inheritance or lineage asymmetry). The current study has shown that the cellular quantum dot fluorescence reduced over time as the particles were diluted by the process of cell division and had the properties of a non-random highly asymmetric event. Asymmetric nanoparticle segregation in the endosomal compartment has major implications on cell-fate determining signaling pathways and could lead to an understanding of the origins of unique proliferation and drug-resistance characteristics within a tumour cell lineage.