Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats.

AIM: To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. METHODS: The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. RESULTS: The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats' retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. CONCLUSION: Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Assessing the bioefficacy of a commercial temephos formulation (Temebate®) for controlling Aedes albopictus larvae in different land use localities in Malaysia.

Temephos is the World Health Organization (WHO) recommended larvicide and is still being utilized worldwide to control larvae of dengue vectors; Aedes aegypti and Aedes albopictus. The efficacy of a commercial temephos product; Temebate® to exterminate the local populations of Ae. albopictus larvae originated from different land use particularly dengue-risk and dengue-free housing localities as well as agrarian localities including oil palm plantations, rubber estates and paddy fields was assessed to verify its bioefficacy in these localities. Field populations of Ae. albopictus larvae were attained via a larval survey at each study locality. Each Ae. albopictus larval population was subjected to a 24-h larval bioassay using Temebate® at operational dosage of 1 mg/L. Almost all Ae. albopictus larval populations demonstrated mortalities between 7.00% and 100.00% by the end of the first 4 h of Temebate® exposure with the resistance ratios between 0.94 and 8.33. After 24 h of Temebate® exposure, all sixteen Ae. albopictus larval populations exhibited increased mortalities with ten of them showing 100% mortalities. These results confirmed the relevance of Temebate® to be continuously used by the residents of these localities as their control efforts against dengue vectors. Nevertheless, Temebate® application by consumers in dengue-risk localities need to be carefully monitored to prevent further development of temephos resistance among Ae. albopictus populations and substantiated with other vector control approaches.

A single targeted gamma-ray irradiation induced an acute modulation of immune cells and related cytokines in EMT6 mouse-bearing tumour model.

BACKGROUND: A complicated interplay between radiation doses, tumour microenvironment (TME), and host immune system is linked to the active participation of immune response. OBJECTIVE: The effects of single targeted 2 Gy and 8 Gy gamma-ray irradiations on the immune cell population (lymphocytes, B-cells, T-cells, neutrophils, eosinophils, and macrophages) in EMT6 mouse-bearing tumour models was investigated. METHODS: The effects of both irradiation doses in early (96 hours) and acute phase (5 to 11 days) post-irradiation on immune parameters were monitored in blood circulation and TME using flow cytometry. Simultaneously, selected cytokines related to immune cells within the TME were measured using multiplex ELISA. RESULTS: A temporary reduction in systemic total white blood count (TWBC) resulted from an early phase (96 hours) of gamma-ray irradiation at 2 Gy and 8 Gy compared to sham control group. No difference was obtained in the acute phase. Neutrophils dominated among other immune cells in TME in sham control group. Eosinophils in TME was significantly increased after 8 Gy treatment in acute phase compared to sham control (p< 0.005). Furthermore, the increment of tumour necrosis (TNF)-α, eotaxin and interleukin (IL)-7 (p< 0.05) in both treatment groups and phases were associated with anti-tumour activities within TME by gamma-ray irradiation. CONCLUSION: The temporary changes in immune cell populations within systemic circulation and TME induced by different doses of gamma-ray irradiation correlated with suppression of several pro-tumorigenic cytokines in mouse-bearing EMT6 tumour models.

Acquired radioresistance in EMT6 mouse mammary carcinoma cell line is mediated by CTLA-4 and PD-1 through JAK/STAT/PI3K pathway.

Cancer recurrence is often associated with the acquisition of radioresistance by cancer tissues due to failure in radiotherapy. The underlying mechanism leading to the development of acquired radioresistance in the EMT6 mouse mammary carcinoma cell line and the potential pathway involved was investigated by comparing differential gene expressions between parental and acquired radioresistance cells. EMT6 cell line was exposed to 2 Gy/per cycle of gamma-ray and the survival fraction between EMT6-treated and parental cells was compared. EMT6RR_MJI (acquired radioresistance) cells was developed after 8 cycles of fractionated irradiation. The development of EMT6RR_MJI cells was confirmed with further irradiation at different doses of gamma-ray, and both the survival fraction and migration rates were measured. Higher survival fraction and migration rates were obtained in EMT6RR_MJI cells after exposure to 4 Gy and 8 Gy gamma-ray irradiations compared to their parental cells. Gene expression between EMT6RR_MJI and parental cells was compared, and 16 genes identified to possess more than tenfold changes were selected and validated using RT-PCR. Out of these genes, 5 were significantly up-regulated i.e., IL-6, PDL-1, AXL, GAS6 and APCDD1. Based on pathway analysis software, the development of acquired radioresistance in EMT6RR_MJI was hypothesized through JAK/STAT/PI3K pathway. Presently, CTLA-4 and PD-1 were determined to be associated with JAK/STAT/PI3K pathway, where both their expressions were significantly increased in EMT6RR_MJI compared to parental cells in the 1st, 4th and 8th cycle of radiation. As a conclusion, the current findings provided a mechanistic platform for the development of acquired radioresistance in EMT6RR_MJI through overexpression of CTLA-4 and PD-1, and novel knowledge on therapeutic targets for recurrent radioresistant cancers.

Hyperparameter Tuning and Pipeline Optimization via Grid Search Method and Tree-Based AutoML in Breast Cancer Prediction.

Automated machine learning (AutoML) has been recognized as a powerful tool to build a system that automates the design and optimizes the model selection machine learning (ML) pipelines. In this study, we present a tree-based pipeline optimization tool (TPOT) as a method for determining ML models with significant performance and less complex breast cancer diagnostic pipelines. Some features of pre-processors and ML models are defined as expression trees and optimal gene programming (GP) pipelines, a stochastic search system. Features of radiomics have been presented as a guide for the ML pipeline selection from the breast cancer data set based on TPOT. Breast cancer data were used in a comparative analysis of the TPOT-generated ML pipelines with the selected ML classifiers, optimized by a grid search approach. The principal component analysis (PCA) random forest (RF) classification was proven to be the most reliable pipeline with the lowest complexity. The TPOT model selection technique exceeded the performance of grid search (GS) optimization. The RF classifier showed an outstanding outcome amongst the models in combination with only two pre-processors, with a precision of 0.83. The grid search optimized for support vector machine (SVM) classifiers generated a difference of 12% in comparison, while the other two classifiers, naïve Bayes (NB) and artificial neural network-multilayer perceptron (ANN-MLP), generated a difference of almost 39%. The method's performance was based on sensitivity, specificity, accuracy, precision, and receiver operating curve (ROC) analysis.

Stability and Reproducibility of Radiomic Features Based Various Segmentation Technique on MR Images of Hepatocellular Carcinoma (HCC).

Hepatocellular carcinoma (HCC) is considered as a complex liver disease and ranked as the eighth-highest mortality rate with a prevalence of 2.4% in Malaysia. Magnetic resonance imaging (MRI) has been acknowledged for its advantages, a gold technique for diagnosing HCC, and yet the false-negative diagnosis from the examinations is inevitable. In this study, 30 MR images from patients diagnosed with HCC is used to evaluate the robustness of semi-automatic segmentation using the flood fill algorithm for quantitative features extraction. The relevant features were extracted from the segmented MR images of HCC. Four types of features extraction were used for this study, which are tumour intensity, shape feature, textural feature and wavelet feature. A total of 662 radiomic features were extracted from manual and semi-automatic segmentation and compared using intra-class relation coefficient (ICC). Radiomic features extracted using semi-automatic segmentation utilized flood filling algorithm from 3D-slicer had significantly higher reproducibility (average ICC = 0.952 ± 0.009, p < 0.05) compared with features extracted from manual segmentation (average ICC = 0.897 ± 0.011, p > 0.05). Moreover, features extracted from semi-automatic segmentation were more robust compared to manual segmentation. This study shows that semi-automatic segmentation from 3D-Slicer is a better alternative to the manual segmentation, as they can produce more robust and reproducible radiomic features.

Development of custom lead shield and strainer for targeted irradiation for mice in the gamma cell chamber.

We presented a development of a custom lead shield and mouse strainer for targeted irradiation from the gamma-cell chamber. This study was divided into two parts i.e., to (i) fabricate the shield and strainer from a lead (Pb) and (ii) optimize the irradiation to the mice-bearing tumour model with 2 and 8 Gy absorbed doses. The lead shielding was fabricated into a cuboid shape with a canal on the top and a hole on the vertical side for the beam path. Respective deliveries doses of 28 and 75 Gy from gamma-cell were used to achieve 2 and 8 Gy absorbed doses at the tumour sites.

An evaluation of novel real-time technology as a tool for measurement of radiobiological and radiation-induced bystander effects.

The xCELLigence real-time cell impedance system uses a non-invasive and label-free method to create a cell index that is a composite measure of cell proliferation. The aim of this study was to evaluate xCELLigence against clonogenic assay (gold standard) for measuring radiobiological effects and radiation-induced bystander effects (RIBE). A radiobiological study was conducted by irradiating EMT6.5, 4T1.2 and NMUMG cell lines with different radiation doses, while a RIBE study was done using transfer of conditioned media (CM) harvested from donor to the same type of recipient cell (EMT6.5, 4T1.2, NMUMG, HACAT and SW48). CM was harvested using two protocols which differed in the dose chosen and the exposure to the recipient cells. Results showed that xCELLigence measured a radiobiological effect which correlated with the clonogenic assay. For the RIBE study, no statistically significant differences were observed between xCELLigence or clonogenic survival in control or recipient cells incubated with CM in protocol one. However, there was a significant increase in cell index slope using CM from EMT-6.5 cells irradiated at 7.5 Gy compared with the control group under the second protocol. No other evidence of RIBE was detected by either xCELLigence or clonogenic assay. In conclusion, xCELLigence methods can measure radiobiological effects and the results correlate with clonogenic assay. We observed a lack of RIBE in all tested cell lines with the clonogenic assay; however, we observed a RIBE effect in EMT6.5 cells under one particular protocol that showed RIBE is cell type dependent, is not universally observed and can be detected in different assays.

An evaluation of dose equivalence between synchrotron microbeam radiation therapy and conventional broad beam radiation using clonogenic and cell impedance assays.

BACKGROUND: High-dose synchrotron microbeam radiation therapy (MRT) has shown the potential to deliver improved outcomes over conventional broadbeam (BB) radiation therapy. To implement synchrotron MRT clinically for cancer treatment, it is necessary to undertake dose equivalence studies to identify MRT doses that give similar outcomes to BB treatments. AIM: To develop an in vitro approach to determine biological dose equivalence between MRT and BB using two different cell-based assays. METHODS: The acute response of tumour and normal cell lines (EMT6.5, 4T1.2, NMuMG, EMT6.5ch, 4T1ch5, SaOS-2) to MRT (50-560 Gy) and BB (1.5-10 Gy) irradiation was investigated using clonogenic and real time cell impedance sensing (RT-CIS)/xCELLigence assays. MRT was performed using a lattice of 25 or 50 µm-wide planar, polychromatic kilovoltage X-ray microbeams with 200 µm peak separation. BB irradiations were performed using a Co60 teletherapy unit or a synchrotron radiation source. BB doses that would generate biological responses similar to MRT were calculated by data interpolation and verified by clonogenic and RT-CIS assays. RESULTS: For a given cell line, MRT equivalent BB doses identified by RT-CIS/xCELLigence were similar to those identified by clonogenic assays. Dose equivalence between MRT and BB were verified in vitro in two cell lines; EMT6.5ch and SaOS-2 by clonogenic assays and RT-CIS/xCELLigence. We found for example, that BB doses of 3.4±0.1 Gy and 4.40±0.04 Gy were radiobiologically equivalent to a peak, microbeam dose of 112 Gy using clonogenic and RT-CIS assays respectively on EMT6.5ch cells. CONCLUSION: Our data provides the first determination of biological dose equivalence between BB and MRT modalities for different cell lines and identifies RT-CIS/xCELLigence assays as a suitable substitute for clonogenic assays. These results will be useful for the safe selection of MRT doses for future veterinary and clinical trials.