Methods: This study investigated the cytotoxic effect of the venom from Trimeresurus purpureomaculatus, the mangrove pit viper (also known as shore pit viper) which is native in Malaysia, across a panel of human cancer cell lines from breast, lung, colon and prostate as well as the corresponding normal cell lines of each tissue.
Results: The venom exhibited dose-dependent cytotoxic activities on all cell lines tested, with median inhibition concentrations (IC50) ranging from 0.42 to 6.98 µg/mL. The venom has a high selectivity index (SI = 14.54) on breast cancer cell line (MCF7), indicating that it is significantly more cytotoxic toward the cancer than to normal cell lines. Furthermore, the venom was fractionated using C18 reversed-phase high-performance liquid chromatography and the anticancer effect of each protein fraction was examined. Fraction 1 that contains a hydrophilic low molecular weight (approximately 7.5 kDa) protein was found to be the most cytotoxic and selective toward the breast cancer cell line (MCF7). The protein was identified using liquid chromatography-tandem mass spectrometry as a venom disintegrin, termed purpureomaculin in this study.
Conclusion: Taken together, the findings revealed the potent and selective cytotoxicity of a disintegrin protein isolated from the Malaysian T. purpureomaculatus venom and suggested its anticancer potential in drug discovery.
OBJECTIVE: To employ machine learning (ML) and stacked ensemble learning (EL) methods in predicting short- and long-term mortality in Asian patients diagnosed with NSTEMI/UA and to identify the associated features, subsequently evaluating these findings against established risk scores.
METHODS: We analyzed data from the National Cardiovascular Disease Database for Malaysia (2006-2019), representing a diverse NSTEMI/UA Asian cohort. Algorithm development utilized in-hospital records of 9,518 patients, 30-day data from 7,133 patients, and 1-year data from 7,031 patients. This study utilized 39 features, including demographic, cardiovascular risk, medication, and clinical features. In the development of the stacked EL model, four base learner algorithms were employed: eXtreme Gradient Boosting (XGB), Support Vector Machine (SVM), Naive Bayes (NB), and Random Forest (RF), with the Generalized Linear Model (GLM) serving as the meta learner. Significant features were chosen and ranked using ML feature importance with backward elimination. The predictive performance of the algorithms was assessed using the area under the curve (AUC) as a metric. Validation of the algorithms was conducted against the TIMI for NSTEMI/UA using a separate validation dataset, and the net reclassification index (NRI) was subsequently determined.
RESULTS: Using both complete and reduced features, the algorithm performance achieved an AUC ranging from 0.73 to 0.89. The top-performing ML algorithm consistently surpassed the TIMI risk score for in-hospital, 30-day, and 1-year predictions (with AUC values of 0.88, 0.88, and 0.81, respectively, all p < 0.001), while the TIMI scores registered significantly lower at 0.55, 0.54, and 0.61. This suggests the TIMI score tends to underestimate patient mortality risk. The net reclassification index (NRI) of the best ML algorithm for NSTEMI/UA patients across these periods yielded an NRI between 40-60% (p < 0.001) relative to the TIMI NSTEMI/UA risk score. Key features identified for both short- and long-term mortality included age, Killip class, heart rate, and Low-Molecular-Weight Heparin (LMWH) administration.
CONCLUSIONS: In a broad multi-ethnic population, ML approaches outperformed conventional TIMI scoring in classifying patients with NSTEMI and UA. ML allows for the precise identification of unique characteristics within individual Asian populations, improving the accuracy of mortality predictions. Continuous development, testing, and validation of these ML algorithms holds the promise of enhanced risk stratification, thereby revolutionizing future management strategies and patient outcomes.
Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.
Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.
Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.