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

Tocotrienol rich fraction supplementation improved lipid profile and oxidative status in healthy older adults: A randomized controlled study

<p>Abstract</p> <p>Background</p> <p>Vitamin E supplements containing tocotrienols are now being recommended for optimum health but its effects are scarcely known. The objective was to determine the effects of Tocotrienol Rich Fraction (TRF) supplementation on lipid profile and oxidative status in healthy older individuals at a dose of 160 mg/day for 6 months.</p> <p>Methods</p> <p>Sixty-two subjects were recruited from two age groups: 35-49 years (n = 31) and above 50 years (n = 31), and randomly assigned to receive either TRF or placebo capsules for six months. Blood samples were obtained at 0, 3^rdand 6^thmonths.</p> <p>Results</p> <p>HDL-cholesterol in the TRF-supplemented group was elevated after 6 months (p < 0.01). Protein carbonyl contents were markedly decreased (p < 0.001), whereas AGE levels were lowered in the > 50 year-old group (p < 0.05). Plasma levels of total vitamin E particularly tocopherols were significantly increased in the TRF-supplemented group after 3 months (p < 0.01). Plasma total tocotrienols were only increased in the > 50 year-old group after receiving 6 months of TRF supplementation. Changes in enzyme activities were only observed in the > 50 year-old group. SOD activity was decreased after 3 (p < 0.05) and 6 (p < 0.05) months of TRF supplementation whereas CAT activity was decreased after 3 (p < 0.01) and 6 (p < 0.05) months in the placebo group. GPx activity was increased at 6 months for both treatment and placebo groups (p < 0.05).</p> <p>Conclusion</p> <p>The observed improvement of plasma cholesterol, AGE and antioxidant vitamin levels as well as the reduced protein damage may indicate a restoration of redox balance after TRF supplementation, particularly in individuals over 50 years of age.</p

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

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

Assessment of radiation effective dose from lung cancer screening pilot project in Institut Kanser Negara: a preliminary finding

The aim of this study is to evaluate effective dose received by participants from Lung Cancer Screening program in Institut Kanser Negara (IKN), Putrajaya. This retrospective study was performed between April 2016 – December 2016 where all scanning acquisition protocols and dose information from forty (40) participants were recorded and investigated. The screening process involves two types of imaging technique, the Dual Energy Subtraction (DES) Chest Xray and Low-Dose Computed Tomography (LDCT) imaging technique. Participant’s effective dose (ED) from DES and MSCT were analysed by using PCXMC (Version 2.0, Finland) and CT-EXPO (Version 2.3, Germany) software, respectively. It was observed that the mean (±SD) value for DES (at 60 kV), DES (at 120 kV) and MSCT examinations were 0.006 ± 0.005 mSv, 0.018 ± 0.005 mSv and 1.558 ± 0.129 mSv, respectively. In a whole, the total cumulative ED values for participants were ranged from 1.376 mSv to 1.986 mSv. It was summarized that both optimized techniques were useful for screening needs and the ED value from this study were lower when compared to other established reference

Estimation of effective dose and organ cancer risk from paediatric computed tomography thorax – Abdomen - Pelvis examinations

Radiation dose exposure in computed tomography (CT) is acknowledged higher when compared to other imaging modalities. The side effects on children have always been a considerable concern. This retrospective study evaluated the dose exposure and organ cancer risk in paediatric patients who underwent CT thorax-abdomen-pelvis (CT TAP) between January 2016 and December 2018. The records of 34 boys and 39 girls (n = 73) were retrieved and divided into three age groups (0–4, 5–9 and 10–12) — the standard protocol for helical scanning setup at 100 kV and 210 mAs. Generally, the CT dose index volume, dose length product and effective dose increased in tandem with the children's age due to higher body mass index. From the youngest to the oldest age group, the liver had the highest cancer risk, especially for boys, which was most likely due to its anatomical location in the scan area. However, from age 5 and above, steep increments were observed in the risk for ovaries, whereas testicles had none. Thus, it is recommended to take precautions when subjecting children to CT TAP, especially prepubescent girls, whose ovaries exposed to almost a thousand times cancer risk per 100,000 procedure. © 2019 Elsevier Lt

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). 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

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)-±, 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

Stability and Reproducibility of Radiomic Features Based on Various Segmentation Techniques on Cervical Cancer DWI-MRI

Cervical cancer is the most common cancer and ranked as 4th in morbidity and mortality among Malaysian women. Currently, Magnetic Resonance Imaging (MRI) is considered as the gold standard imaging modality for tumours with a stage higher than IB2, due to its superiority in diagnostic assessment of tumour infiltration with excellent soft-tissue contrast. In this research, the robustness of semi-automatic segmentation has been evaluated using a flood-fill algorithm for quantitative feature extraction, using 30 diffusion weighted MRI images (DWI-MRI) of cervical cancer patients. The relevant features were extracted from DWI-MRI segmented images of cervical cancer. First order statistics, shape features, and textural features were extracted and analysed. The intra-class relation coefficient (ICC) was used to compare 662 radiomic features extracted from manual and semi-automatic segmentations. Notably, the features extracted from the semi-automatic segmentation and flood filling algorithm (average ICC = 0.952 0.009, p &gt; 0.05) were significantly higher than the manual extracted features (average ICC = 0.897 0.011, p &gt; 0.05). Henceforth, we demonstrate that the semi-automatic segmentation is slightly expanded to manual segmentation as it produces more robust and reproducible radiomic features

Comparison of cancer stem cell enrichment between spheroids derived from single-cell and multicellular aggregate cultures

Introduction: Cancer stem cells (CSCs) represent a distinct group of cells within cancerous tissue that possess the ability to initiate tumorigenesis and exhibit potency, self-renewal, and drug resistance. The study of CSCs often encounters challenges in obtaining these cells of interest or generating a sufficient quantity for downstream analysis. Nevertheless, it is feasible to enrich CSCs in vitro by subjecting them to conditions that stimulate their CSC properties, such as prolonged exposure to drugs or radiation, or by promoting their self-renewal capability through spheroid culture. Spheroids are a specific type of cell culture that organizes cells into a three-dimensional structure, closely mimicking the in vivo environment. These spheroids consist of a heterogeneous cell population, including CSCs or tumor-propagating cells responsible for tumor growth and maintenance. In our study, we cultured spheroids derived from single cells as well as multicellular aggregates to enrich CSCs based on their self-renewal capability and the structural organization provided by the three-dimensional context. Methods: Comparing the spheroid cultures with the parental adherent monolayer cells, we observed higher expression of CSC markers, pluripotent genes, and adipogenic differentiation in both multicellular spheroids (MCS) and single cell-derived spheroids (SCDS) of the two tested cell lines. Results: The spheroids exhibited progressive growth in size throughout the culture period. When comparing the two methods, SCDS demonstrated greater expression of surface markers and all three pluripotent genes associated with CSCs. Furthermore, when assessing drug resistance potential and the expression of the ABCG2 drug efflux gene, only 5637 SCDS displayed increased resistance to cisplatin and upregulation of ABCG2. Conclusion: In conclusion, both the MCS and SCDS methods effectively enriched the population of bladder CSCs in the 5637 and HT-1376 bladder cancer cell lines. However, the SCDS method demonstrated a higher upregulation of CSC markers and pluripotent gene expression compared to MCS. It is worth noting that spheroid culture and CSC enrichment are not mutually exclusive and can coexist with increased chemotherapy resistance and upregulation of ABCG2 drug efflux gene expression. Moreover, the drug efflux capability may vary depending on the specific cell line and clonal lineage. These strategies can serve as valuable models for CSC enrichment, the study of cancer cell behavior, disease modeling, and personalized chemotherapy investigations