Single-hit Model Research Articles

Newly discovered letter: why Muller failed to cite the negative mouse mutation findings of Snell, preserving his chances to receive the Nobel Prize.

A recently acquired letter between Hermann Muller and his wife (March 21, 1933) reveals that Muller had learned that he had been nominated for the Nobel Prize in 1932 with about 1/3 of the total votes being supportive. Muller was hopeful that over time sufficient votes would lead to receiving the award. The knowledge of Muller on this matter and its timing provide a likely explanation why Muller never cited the negative mouse mutation findings of George Snell, performed under Muller's direction during that time period. This action of Muller, along with the failure of Snell to promote his discovery, greatly reduced the chances that those findings would complicate his attempt to garner support for his LNT single-hit model and its application to hereditary and cancer risk assessment. It also helped Muller achieve the Nobel Prize, allowing him the necessary international visibility to promote his ideologically driven ionizing radiation-related LNT-based paradigm.

Dynamics of local gene regulations in synovial fluid leukocytes from horses with lipopolysaccharide-induced arthritis

The role of resident cells such a synoviocytes and chondrocytes in intra-articular inflammation is well-characterized, however the in vivo gene expression patterns of cells (predominantly leukocytes) in the synovial fluid (SF) of an inflamed joint have never previously been investigated. The aim of this study was to investigate gene expression in SF leukocytes from the inflamed joint cavity after intra-articular lipopolysaccharide (LPS) injection in horses to improve our understanding of the temporal regulation of the intra-articular inflammatory response. Gene expression was investigated in SF samples available from six horses 2, 4, 8 16 and 24 h after experimental induction of inflammation in the radiocarpal joint by lipopolysaccharide (LPS) injection. Leukocytic expression of 43 inflammation-related genes was studied using microfluidic high throughput qPCR (Fluidigm®). Expression of 26 genes changed significantly over the 24 h study period, including pro- and anti-inflammatory genes such as interleukin (IL)1, IL6, tumor necrosis factor (TNF), cyclooxygenase 2 (COX2), IL1 receptor antagonist (IL1RN), IL10, and superoxide dismutase 2 (SOD2), chemokine genes, apoptosis-related genes, and genes related to cartilage turnover (matrix metalloproteinase 8 and tissue inhibitor of metalloproteinase 1). The inflammatory responses appeared to be regulated, as an early increase (at 2 h) in expression of the pro-inflammatory genes IL1, IL6, TNF and COX2 was rapidly followed by increased expression (at 4 h) of several anti-inflammatory genes (IL10, IL1RN and SOD2). Similarly, both pro- and anti-apoptotic gene expression as well as expression of chondrodegenerative and chondroprotective genes were activated in SF leukocytes. Thus, the inflammatory response in leukocytes infiltrating the joint in the acute stage of arthritis was well orchestrated in this single-hit LPS-induced arthritis model. This study is the first to describe gene expression patterns in SF-derived leukocytes in vivo during severe joint inflammation, and the results thus expand our knowledge of basic inflammatory mechanisms in the early local response in an inflamed joint.

ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-\u03b2 receptor II in breast cancer

BackgroundRadiotherapy is a conventional and effective local treatment for breast cancer. However, residual or recurrent tumors appears frequently because of radioresistance. Novel predictive marker and the potential therapeutic targets of breast cancer radioresistance needs to be investigated.MethodsIn this study, we screened all 10 asparagine-linked glycosylation (ALG) members in breast cancer patients’ samples by RT-PCR. Cell viability after irradiation (IR) was determined by CCK-8 assay and flow cytometry. The radiosensitivity of cell lines with different ALG3 expression was determined with the colony formation assay by fitting the multi-target single hit model to the surviving fractions. Cancer stem-like traits were assessed by RT-PCR, Western blot, and flow cytometry. The mechanisms of ALG3 influencing radiosensitivity was detected by Western blot and immunoprecipitation. And the effect of ALG3 on tumor growth after IR was verified in an orthotopic xenograft tumor models. The association of ALG3 with prognosis of breast cancer patients was confirmed by immunohistochemistry.ResultsALG3 was the most significantly overexpressing gene among ALG family in radioresistant breast cancer tissue. Overexpression of ALG3 predicted poor clinicopathological characteristics and overall survival (OS), and early local recurrence-free survival (LRFS) in breast cancer patients. Upregulating ALG3 enhanced radioresistance and cancer stemness in vitro and in vivo. Conversely, silencing ALG3 increased the radiosensitivity and repressed cancer stemness in vitro, and more importantly inhibition of ALG3 effectively increased the radiosensitivity of breast cancer cells in vivo. Mechanistically, our results further revealed ALG3 promoted radioresistance and cancer stemness by inducing glycosylation of TGF-β receptor II (TGFBR2). Importantly, both attenuation of glycosylation using tunicamycin and inhibition of TGFBR2 using LY2109761 differentially abrogated the stimulatory effect of ALG3 overexpression on cancer stemness and radioresistance. Finally, our findings showed that radiation played an important role in preventing early recurrence in breast cancer patients with low ALG3 levels, but it had limited efficacy in ALG3-overexpressing breast cancer patients.ConclusionOur results suggest that ALG3 may serve as a potential radiosensitive marker, and an effective target to decrease radioresistance by regulating glycosylation of TGFBR2 in breast cancer. For patients with low ALG3 levels, radiation remains an effective mainstay therapy to prevent early recurrence in breast cancer.

The significance of the failed historical foundation of linear non-threshold model for cancer risk assessment

The linear non-threshold (LNT) single-hit (SH) dose response model for cancer risk assessment is assessed with respect to its historical foundations. This paper examines and summarises how mistakes, ideological biases, and scientific misconduct by key scientists affected the acceptance, validity, and application of the LNT single-hit model for cancer risk assessment. This analysis concludes that the LNT single-hit model was inappropriately adopted for governmental risk assessment, regulatory policy and practices, and for risk communication.

The significance of the failed historical foundation of linear non-threshold model for cancer risk assessment

The linear non-threshold (LNT) single-hit (SH) dose response model for cancer risk assessment is assessed with respect to its historical foundations. This paper examines and summarises how mistakes, ideological biases, and scientific misconduct by key scientists affected the acceptance, validity, and application of the LNT single-hit model for cancer risk assessment. This analysis concludes that the LNT single-hit model was inappropriately adopted for governmental risk assessment, regulatory policy and practices, and for risk communication.

Radiosensitization effects of curcumin plus cisplatin on non‑small cell lung cancer A549 cells

The aim of the present study was to determine the radiosensitization effect of the combination of curcumin and cisplatin on non-small cell lung cancer (NSCLC) A549 cells. Cell viability was analyzed using the MTT assay following treatment with different concentrations of curcumin and cisplatin for 24~72 h. Survival fraction (SF) value of the treatment groups (single irradiation, curcumin + irradiation, cisplatin + irradiation, and curcumin + cisplatin + irradiation) treated with different doses of X-ray radiation were evaluated using colony formation assay, according to a multi-target single-hit model. Migration and invasion as well as the levels of epidermal growth factor receptor (EGFR) protein following 24 h were detected by scratch wound assay, Matrigel assay and western blot analysis, respectively. The results of the present study demonstrated that the viability of the cells decreased after being treated by curcumin, and the inhibitory effect was dose and time-dependent as the concentration of curcumin increased from 10 to 200 µmol/l (P<0.05). SF value was lower in the curcumin + cisplatin + irradiation group compared with the other three treatment groups at 2~10 Gy. Furthermore, SF value was lower in the curcumin + irradiation group at 4~10 Gy. The SF value was also lower in the cisplatin + irradiation group at 2~10 Gy compared with the single irradiation group (P<0.05). The sensitization enhancement ratios in the curcumin + irradiation, cisplatin + irradiation, and curcumin + cisplatin + irradiation groups were 1.24, 1.31 and 1.96, respectively. The migration distance, the number of cells invaded through the transmembrane, and the level of EGFR protein in four treatment groups were the highest in the single irradiation group, compared with the other three treatment groups (P<0.05). Furthermore, the radiosensitization effects of curcumin and cisplatin on NSCLC A549 cells, which include inhibition of proliferation, migration and invasion, may be associated with the inhibition of the EGFR-associated signaling pathway.

LncRNA CCAT1 enhances radiosensitivity of human pancreatic cancer cells PANC-1 by targeting miR-130b-3p

Objective To investigate the effect of lncRNA CCAT1 and miR-130b-3p on the radiosensitivity of human pancreatic cancer cells PANC-1. Methods Real-time PCR was used to detect the relative expression levels of CCAT1 and miR-130b-3p in pancreatic cancer tissues and cell lines including PANC-1 cells irradiated with 2 Gy X-rays. After silencing CCAT1 and/or inhibiting miR-130b-3p expression, cell apoptosis rate, Caspase 3 activity and cell survival were detected by flow cytometry, Caspase 3 activity detection kit and colony formation assay, respectively. Cell survival curve was stimulated by the multi-target single-hit model. Based on the starBase v2.0 online analysis, the luciferase reporter gene assay, RNA-binding protein immunoprecipitation assay (RIP) and Real-time PCR assay were applied to verify the relationship between CCAT1 and miR-130b-3p. Results CCAT1 expression was up-regulated (t=6.322-8.555, P<0.05), but miR-130b-3p expression was down-regulated (t=3.950-18.795, P<0.05) in the radiation-resistant pancreatic cancer tissues, pancreatic cancer cell lines and 2 Gy-irradiated PANC-1 cells. When the CCAT1 silenced PANC-1 cells were irradiated with 2 Gy, cell survival fraction decreased (t=2.929, 5.047, 5.234, 5.125, P<0.05), apoptosis rate and Caspase 3 activity increased (t=6.953, 6.836, P<0.05). CCAT1 could selectively regulate miR-130b-3p expression. Inhibition of miR-130b-3p expression could enhance PANC-1 cell survival (t=4.564, 6.736, 8.656, P<0.05), but reduced apoptosis rate (t=5.234, P<0.05) and Caspase 3 activity (t=10.440, P<0.05). Conclusions Silencing CCAT1 promotes the expression of miR-130b-3p and enhances radiosensitivity of PANC-1 cells. Key words: lncRNA CCAT1; miR-130b-3p; Pancreatic cancer; Radiosensitivity

EPA transparency proposal: testimony of Edward J. Calabrese, Ph.D, October 3, 2018.

The historical foundations of cancer risk assessment and its adoption by the US EPA in the mid 1970s were based on the discovery of X-ray-induced gene mutations by Hermann J. Muller, its transformation into the linear nonthreshold (LNT) single-hit theory and the recommendations of the model by the US National Academy of Sciences, Biological Effects of Atomic/Ionizing Radiation, Genetics Panels in 1956 and 1972. This testimony summarizes substantial recent revelations which profoundly challenge the use of LNT as a default in cancer risk assessment, showing multiple significant scientific errors and incorrect interpretations, mixed with deliberate misrepresentation of the scientific record by leading ideologically motivated radiation geneticists. These novel historical and scientific findings demonstrate that the scientific foundations of the LNT single-hit model were seriously flawed and should not have been adopted for cancer risk assessment. The testimony supports the recommendation by the EPA to move away from the use of the LNT as a default in cancer risk assessment and to the formal consideration of alternative dose response models such as the threshold, hormetic and other non-linear modeling approaches.

Validation of Quantitative Microbial Risk Assessment Using Epidemiological Data from Outbreaks of Waterborne Gastrointestinal Disease.

The assumptions underlying quantitative microbial risk assessment (QMRA) are simple and biologically plausible, but QMRA predictions have never been validated for many pathogens. The objective of this study was to validate QMRA predictions against epidemiological measurements from outbreaks of waterborne gastrointestinal disease. I screened 2,000 papers and identified 12 outbreaks with the necessary data: disease rates measured using epidemiological methods and pathogen concentrations measured in the source water. Eight of the 12 outbreaks were caused by Cryptosporidium, three by Giardia, and one by norovirus. Disease rates varied from 5.5 × 10-6 to 1.1 × 10-2 cases/person-day, and reported pathogen concentrations varied from 1.2 × 10-4 to 8.6 × 102 per liter. I used these concentrations with single-hit dose-response models for all three pathogens to conduct QMRA, producing both point and interval predictions of disease rates for each outbreak. Comparison of QMRA predictions to epidemiological measurements showed good agreement; interval predictions contained measured disease rates for 9 of 12 outbreaks, with point predictions off by factors of 1.0-120 (median = 4.8). Furthermore, 11 outbreaks occurred at mean doses of less than 1 pathogen per exposure. Measured disease rates for these outbreaks were clearly consistent with a single-hit model, and not with a "two-hit" threshold model. These results demonstrate the validity of QMRA for predicting disease rates due to Cryptosporidium and Giardia.

Experimental investigation of GafChromic® EBT3 intrinsic energy dependence with kilovoltage x rays, 137 Cs, and 60 Co.

To determine experimentally the intrinsic energy response, kbq , of EBT3 GafChromic® radiochromic film with kilovoltage x rays, 137 Cs, and 60 Co in therapeutic and diagnostic dose ranges through direct measurement with an accompanying mathematical approach to describe the physical processes involved. The EBT3 film was irradiated with known doses using 60 Co, 137 Cs, and 13 NIST-matched kilovoltage x-ray beams. Seven dose levels, ranging from 57 to 7002mGy, were chosen for this work. Monte Carlo methods were used to convert air-kerma rates to dose rates to the film active layer for each energy. A total of 738 film dosimeters, each measuring (1.2×1.2) cm2 , were cut from three film sheets out of the same lot of the latest version of EBT3 film, to allow for multiple dosimeters to be irradiated by each target dose and beam quality as well as unirradiated dosimeters to be used as controls. Net change in optical density in excess of the unirradiated controls was measured using the UWMRRC Laser Densitometry System (LDS). The dosimeter intrinsic energy response, kbq , for each dose level was determined relative to 60 Co, as the ratio of dosimeter response to each beam quality relative to the absorbed dose to the film active volume at the same dose level. A simplified, single-hit mathematical model was used to derive a single-free-parameter, β, which is a proportionality constant that is dependent on beam quality and describes the microdosimetric interactions within the active layer of film. The response of β for each beam quality relative to 60 Co was also determined. kbq was determined for a wide range of doses and energies. The results show a unique variation of kbq as a function of energy, and agree well with results from other investigations. There was no measurable dose dependence for kbq within the 500-7002mGy range outside of the expanded measurement uncertainty of 3.65% (k=2). For doses less than 500mGy, the signal-to-noise ratio was too low to determine kbq accurately. The single-free-parameter, β, fit calculations derived from the single-hit model show a correlation with kbq that suggests that β, at least in part, characterizes the microdosimetric interactions that determine kbq . For the beam qualities investigated, a single energy-dependent kbq correction can be used for doses between 500 and 7002mGy. Using the single-hit model with the single-free-parameter fit to solve for β shows promise in the determination of the intrinsic energy response of film, with β being the mathematical analog of the measured kbq .

The Mistaken Birth and Adoption of LNT: An Abridged Version.

The historical foundations of cancer risk assessment were based on the discovery of X-ray-induced gene mutations by Hermann J. Muller, its transformation into the linear nonthreshold (LNT) single-hit theory, the recommendation of the model by the US National Academy of Sciences, Biological Effects of Atomic Radiation I, Genetics Panel in 1956, and subsequent widespread adoption by regulatory agencies worldwide. This article summarizes substantial recent historical revelations of this history, which profoundly challenge the standard and widely acceptable history of cancer risk assessment, showing multiple significant scientific errors and incorrect interpretations, mixed with deliberate misrepresentation of the scientific record by leading ideologically motivated radiation geneticists. These novel historical findings demonstrate that the scientific foundations of the LNT single-hit model were seriously flawed and should not have been adopted for cancer risk assessment.

Effect of platycodin D on radiosensitivity of human hepatoma cell line and related mechanisms of action

Objective: To investigate the effect of platycodin D on the radiosensitivity of human hepatoma cell lines HepG2 and SMMC-7721 and related mechanisms of action. Methods: MTT assay was used to analyze the effect of different concentrations of platycodin D with different treatment times on cell viability. The cells were pretreated with 5 μg/ml platycodin D for 24 hours followed by X-ray irradiation at different radiation doses. Colony-forming assay was used to measure the radiosensitizing effect of platycodin D on cells. The quasi-threshold dose (Dq), mean lethal dose (Do), extrapolation number (N), sensitizer enhancement ratio (SER), and survival fraction (SF) at different radiation doses were calculated, and the multi-target single-hit model was used to fit the cell survival curve according to the formula SF = l-(l-e(-D/D0))N. Flow cytometry was used to investigate the distribution of cell cycle, and Western blotting was used to measure the changes in the protein expression of phosphorylated phosphatidylinositol 3'-kinase (pPI3K), phosphorylated protein kinase (pAkt), nuclear factor-κB (NF-κB), and phosphorylated nuclear factor inhibiting protein (pIκBα). A one-way analysis of variance, the t-test, or the least significant difference test was used for statistical analysis based on the type of data. Results: Platycodin D reduced the viability of HepG2 and SMMC-7721 cells in a dose-dependent manner; the IC50 value for HepG2 cells was 24.2 ± 0.61 μg/ml at 24 hours and 7.68 ± 0.46 μg/ml at 48 hours, and that for SMMC-7721 cells was 23.8 ± 0.57 μg/ml at 24 hours and 8.63 ± 0.86 μg/mL at 48 hours. After the combined treatment with platycodin D and irradiation, there were significant reductions in Dq (P = 0.002), Do (P = 0.002), and N value (P = 0.003), the survival curve markedly shifted to the left, and SER was 1.347 ± 0.04 in HepG2 cells and 1.418 ± 0.05 in SMMC-7721 cells. In addition, platycodin D significantly inhibited the increase in the proportion of cells in G2/M phase, the increases in the protein expression of pPI3k (P = 0.002), pAkt (P = 0.003), and NF-κB (P = 0.002), and the reduction in the protein expression of pIκBα (P = 0.003). Conclusion: Platycodin D can increase the radiosensitivity of HepG2 or SMMC-7721 cells, possibly by enhancing the growth inhibition effect of irradiation and inhibiting the activation of the PI3k/Akt and NF-κB pathways.

Modeling the dose dependence of the vis-absorption spectrum of EBT3 GafChromic™ films.

The aim of this work was to model the dose dependence of the darkening of GafChromic™ EBT3 films by combining the optical properties of the polydiacetylene polymer phases, and a modified version of the single-hit model, which will take the stick-like shape of the monomer microcrystals into account. Second, a comparison is made between the quantification of the film darkening by flatbed scanning and by UV-vis absorption spectroscopy. GafChromicTM EBT3 films were irradiated with a 6 MV photon beam at dose levels between 0 and 50 Gy. The radiation-induced darkening of the films is quantified by a flatbed scanner, and by UV-vis absorption spectroscopy in the wavelength range of 220-750 nm. From the UV-vis absorption spectra, the contribution of each polymer phase to the absorbance was deduced. Next, the dose dependence of the polymer content is described by a modified single-hit model where the size distribution of polymerizable centers is approximated by way of the size distribution of the monomer microcrystals in the film. The absorption properties of the film can be accurately quantified by UV-vis spectroscopy for dose levels between 0 and 10 Gy. Over 10 Gy, the absorption spectrum saturates due to the limited sensitivity of the spectrometer. The modified single-hit model was successful in describing the increasing polymer concentration with radiation dose, using a log-normal distribution for the length of the stick-like monomer microcrystals. The dose dependence of the polymer content, deduced from the UV-vis absorption spectrum, differs from that of the flatbed scanning method and is more sensitive to changes in dose. The dose dependence of the polymer concentration can be modeled by taking into account the distribution of active centers using the microstructure of the active layer for dose levels between 0 and 10 Gy. The dissimilar dose dependence of the polymer concentration and the absorbance must be accounted for when modeling darkening from the kinetics of the photopolymerization reaction.

Shape-Dependent Radiosensitization Effect of Gold Nanostructures in Cancer Radiotherapy: Comparison of Gold Nanoparticles, Nanospikes, and Nanorods.

The shape effect of gold (Au) nanomaterials on the efficiency of cancer radiotherapy has not been fully elucidated. To address this issue, Au nanomaterials with different shapes but similar average size (∼50 nm) including spherical gold nanoparticles (GNPs), gold nanospikes (GNSs), and gold nanorods (GNRs) were synthesized and functionalized with poly(ethylene glycol) (PEG) molecules. Although all of these Au nanostructures were coated with the same PEG molecules, their cellular uptake behavior differed significantly. The GNPs showed the highest cellular responses as compared to the GNSs and the GNRs (based on the same gold mass) after incubation with KB cancer cells for 24 h. The cellular uptake in cells increased in the order of GNPs > GNSs > GNRs. Our comparative studies indicated that all of these PEGylated Au nanostructures could induce enhanced cancer cell-killing rates more or less upon X-ray irradiation. The sensitization enhancement ratios (SERs) calculated by a multitarget single-hit model were 1.62, 1.37, and 1.21 corresponding to the treatments of GNPs, GNSs, and GNRs, respectively, demonstrating that the GNPs showed a higher anticancer efficiency than both GNSs and GNRs upon X-ray irradiation. Almost the same values were obtained by dividing the SERs of the three types of Au nanomaterials by their corresponding cellular uptake amounts, indicating that the higher SER of GNPs was due to their much higher cellular uptake efficiency. The above results indicated that the radiation enhancement effects were determined by the amount of the internalized gold atoms. Therefore, to achieve a strong radiosensitization effect in cancer radiotherapy, it is necessary to use Au-based nanomaterials with a high cellular internalization. Further studies on the radiosensitization mechanisms demonstrated that ROS generation and cell cycle redistribution induced by Au nanostructures played essential roles in enhancing radiosensitization. Taken together, our results indicated that the shape of Au-based nanomaterials had a significant influence on cancer radiotherapy. The present work may provide important guidance for the design and use of Au nanostructures in cancer radiotherapy.

Dose-response relationships for environmentally mediated infectious disease transmission models.

Environmentally mediated infectious disease transmission models provide a mechanistic approach to examining environmental interventions for outbreaks, such as water treatment or surface decontamination. The shift from the classical SIR framework to one incorporating the environment requires codifying the relationship between exposure to environmental pathogens and infection, i.e. the dose–response relationship. Much of the work characterizing the functional forms of dose–response relationships has used statistical fit to experimental data. However, there has been little research examining the consequences of the choice of functional form in the context of transmission dynamics. To this end, we identify four properties of dose–response functions that should be considered when selecting a functional form: low-dose linearity, scalability, concavity, and whether it is a single-hit model. We find that i) middle- and high-dose data do not constrain the low-dose response, and different dose–response forms that are equally plausible given the data can lead to significant differences in simulated outbreak dynamics; ii) the choice of how to aggregate continuous exposure into discrete doses can impact the modeled force of infection; iii) low-dose linear, concave functions allow the basic reproduction number to control global dynamics; and iv) identifiability analysis offers a way to manage multiple sources of uncertainty and leverage environmental monitoring to make inference about infectivity. By applying an environmentally mediated infectious disease model to the 1993 Milwaukee Cryptosporidium outbreak, we demonstrate that environmental monitoring allows for inference regarding the infectivity of the pathogen and thus improves our ability to identify outbreak characteristics such as pathogen strain.

RAD-ADAPT: Software for modelling clonogenic assay data in radiation biology

We present a comprehensive software program, RAD-ADAPT, for the quantitative analysis of clonogenic assays in radiation biology. Two commonly used models for clonogenic assay analysis, the linear-quadratic model and single-hit multi-target model, are included in the software. RAD-ADAPT uses maximum likelihood estimation method to obtain parameter estimates with the assumption that cell colony count data follow a Poisson distribution. The program has an intuitive interface, generates model prediction plots, tabulates model parameter estimates, and allows automatic statistical comparison of parameters between different groups. The RAD-ADAPT interface is written using the statistical software R and the underlying computations are accomplished by the ADAPT software system for pharmacokinetic/pharmacodynamic systems analysis. The use of RAD-ADAPT is demonstrated using an example that examines the impact of pharmacologic ATM and ATR kinase inhibition on human lung cancer cell line A549 after ionizing radiation.

Abstract 2384: High-complexity neutral genomic barcoding technology reveals extensive clonal dynamics in multiple human cancer model systems

Abstract Increasing evidence of extensive intratumoral heterogeneity, along with advances in high-throughput in vivo functional genetic screening technologies, have together highlighted the need to observe growth in cancer models at the clonal level. To address this, we have designed, constructed and validated multiple high-diversity lentivirally delivered barcode libraries, which utilize next-gen sequencing technology to read out millions of clones in heterogeneous cancer populations. These libraries can be used to address a multitude of biological questions in many cancer model systems. To date, we have completed, sequenced and analyzed three different types of barcoding applications to explore clonal dynamics in a variety of human cancer models. First, we tested the serial limiting dilution analysis (LDA) assay for tumor initiating cells (TICs) in patient-derived colon tumor models. In the LDA assay, a range of cell dilutions, down to a single cell, are transplanted into immunocompromised mice. The TIC frequency is calculated using a single-hit model from the proportion of tumors established at each dose. However, quantifying minimum cell numbers required for tumor formation does not reveal the actual diversity of clonal contribution during tumour engraftment and progression. In fact, recent research suggests that the single-hit model, which assumes a static hierarchy of intrinsically determined initiating cells, may not be biologically relevant as some clones may have the potential to form or contribute to a tumor only in specific environment, or in cooperation with another clone. Our barcoded, serial LDAs show interesting and informative patterns of clonal dynamics. Second, we have performed clonal lineage tracing on established human cancer cell lines in vitro compared to in vivo. Our findings demonstrate the presence of TICs in standard cell lines and serve as precursors to future in vivo genetic screens by defining upper limits of library size. Finally, we used a patient-derived xenograft model of brain tumor metastasis to investigate the presence of pre-existing metastatic clones with site-specific homing abilities. In summary, given the extensive heterogeneity present in cancer models, we propose the use of high-complexity barcoding technology to validate novel cancer targets in xenograft models, including LDAs and metastasis models. Furthermore, we suggest the use of barcodes for careful optimization of the system before genetic screens in animal models. Citation Format: Allison ML Nixon, Kevin R. Brown, Jennifer Haynes, Laura K. Donovan, Michael D. Taylor, Catherine W. O’Brien, Jason Moffat. High-complexity neutral genomic barcoding technology reveals extensive clonal dynamics in multiple human cancer model systems. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2384.

Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment.

ObjectiveThis study aimed to investigate the impact of S100A4-small interfering RNA (S100A4-siRNA) on apoptosis and enhanced radiosensitivity in non-small-cell lung cancer (A549) cells. We also explored the mechanisms of radiosensitization and identified a new target to enhance radiosensitivity and gene therapy for non-small-cell lung cancer.MethodsRNA interference is a powerful tool for gene silencing. In this study, we constructed an effective siRNA to knock down S100A4. A549 cells were randomly divided into three groups: blank, negative control, and S100A4-siRNA. To investigate the effect of S100A4-siRNA, the expression of S100A4, E-cadherin, and p53 proteins and their messenger RNA (mRNA) was detected by Western blot and quantitative real-time polymerase chain reaction. Transwell chambers were used to assess cell invasion. Cell cycle and apoptosis were analyzed by flow cytometry. Radiosensitivity was determined by colony formation ability.ResultsOur results demonstrate that S100A4-siRNA effectively silenced the S100A4 gene. When siRNA against S100A4 was used, S100A4 protein expression was downregulated, whereas the expressions of E-cadherin and p53 were upregulated. In addition, a clear reduction in S100A4 mRNA levels was noted compared with the blank and negative control groups, whereas E-cadherin and p53 mRNA levels increased. Transfection with S100A4-siRNA significantly reduced the invasiveness of A549 cells. S100A4 silencing induced immediate G2/M arrest in cell cycle studies and increased apoptosis rates in A549 cells. In clonogenic assays, we used a multitarget, single-hit model to detect radiosensitivity after S100A4 knockdown. All parameters (D0, Dq, α, β) indicated that the downregulation of S100A4 enhanced radiosensitivity in A549 cells. Furthermore, S100A4-siRNA upregulated p53 expression, suggesting that S100A4 may promote A549 cell proliferation, invasion, and metastasis by regulating the expression of other proteins. Therefore, siRNA-directed S100A4 knockdown may represent a viable clinical therapy for lung cancer.ConclusionS100A4 downregulation potentially enhances the sensitivity of human A549 cells to radiotherapy.

A Generalized QMRA Beta-Poisson Dose-Response Model.

Quantitative microbial risk assessment (QMRA) is widely accepted for characterizing the microbial risks associated with food, water, and wastewater. Single-hit dose-response models are the most commonly used dose-response models in QMRA. Denoting PI(d) as the probability of infection at a given mean dose d, a three-parameter generalized QMRA beta-Poisson dose-response model, PI(d|α,β,r*), is proposed in which the minimum number of organisms required for causing infection, Kmin , is not fixed, but a random variable following a geometric distribution with parameter 0<r*≤1. The single-hit beta-Poisson model, PI(d|α,β), is a special case of the generalized model with Kmin = 1 (which implies r*=1). The generalized beta-Poisson model is based on a conceptual model with greater detail in the dose-response mechanism. Since a maximum likelihood solution is not easily available, a likelihood-free approximate Bayesian computation (ABC) algorithm is employed for parameter estimation. By fitting the generalized model to four experimental data sets from the literature, this study reveals that the posterior median r* estimates produced fall short of meeting the required condition of r* = 1 for single-hit assumption. However, three out of four data sets fitted by the generalized models could not achieve an improvement in goodness of fit. These combined results imply that, at least in some cases, a single-hit assumption for characterizing the dose-response process may not be appropriate, but that the more complex models may be difficult to support especially if the sample size is small. The three-parameter generalized model provides a possibility to investigate the mechanism of a dose-response process in greater detail than is possible under a single-hit model.

Dual hit lipopolysaccharide & oleic acid combination induced rat model of acute lung injury/acute respiratory distress syndrome.

Background & objectives:Despite advances in therapy and overall medical care, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) management remains a problem. Hence the objective of this study was to develop a rat model that mimics human ALI/ARDS.Methods:Four groups of Wistar rats, 48 per group were treated with (i) intratracheal (IT) lipopolysaccharide (LPS) (5 mg/kg) dissolved in normal saline (NS), (ii) intravenous (iv) oleic acid (OA) (250 μl/kg) suspension in bovine serum albumin (BSA), (iii) dual hit: IT LPS (2 mg/kg) dissolved in NS and iv OA (100 μl/kg) and (iv) control group: IT NS and iv BSA. From each group at set periods of time various investigations like chest X-rays, respiratory rate (RR), tidal volume (TV), total cell count, differential cell count, total protein count and cytokine levels in bronchoalveolar lavage fluid (BALF), lung wet/dry weight ratio and histopathological examination were done.Results:It was noted that the respiratory rate, and tumour necrosis factor-α (TNF-α) levels were significantly higher at 4 h in the dual hit group as compared to LPS, OA and control groups. Interleukin-6 (IL-6) levels were significantly higher in the dual hit group as compared to LPS at 8 and 24 h, OA at 8 h and control (at all time intervals) group. IL-1β levels were significantly higher in LPS and dual hit groups at all time intervals, but not in OA and control groups. The injury induced in dual hit group was earlier and more sustained as compared to LPS and OA alone.Interpretation & conclusions:The lung pathology and changes in respiration functions produced by the dual hit model were closer to the diagnostic criteria of ALI/ARDS in terms of clinical manifestations and pulmonary injury and the injury persisted longer as compared to LPS and OA single hit model. Therefore, the ARDS model produced by the dual hit method was closer to the diagnostic criteria of ARDS in terms of clinical manifestations and pulmonary injury.