Matrix Attachment Regions Enhance Transgene Expression by Manipulating Position-Dependent Effects in Stably Transfected CHO-K1 Cells.

We previously found that the position of matrix attachment regions (MARs) within the vector significantly affects its ability to enhance transgenic expression in the recombinant protein production. This study aims to systematically investigate the position-dependent impacts of MAR on transgene expression. We observed a significant increase in enhanced green fluores-cent protein (eGFP) expression levels in stably transfected CHO-K1 cells with either MAR 1-68 or MAR X-29 when MARs located upstream of the promoter. This increase was especially evident with MAR flanked the expression cassette. Concurrently, a substantial increase was observed in the percentage of eGFP-expressing cells, with 97.8% and 96.0% in MAR-containing constructs versus 73.7% in MAR-absent constructs. Further analysis of erythropoietin (EPO) expression revealed that constructs with flanking MARs induced the highest EPO productivity. Bioinformatics analysis revealed that certain specific transcription factors are important in modulating the transcription process. In conclusion, vectors harboring both MARs around the expression cassette constitute an optimal construct for enhanced recom-binant protein production in CHO-K1 cells. This insight underscores the importance of strate-gic MAR incorporation in vector design for optimized recombinant protein expression.

Recent advances of engineered oncolytic viruses-based combination therapy for liver cancer.

Liver cancer is a major malignant tumor, which seriously threatens human health and increases the economic burden on patients. At present, gene therapy has been comprehensively studied as an excellent therapeutic measure in liver cancer treatment. Oncolytic virus (OV) is a kind of virus that can specifically infect and kill tumor cells. After being modified by genetic engineering, the specificity of OV infection to tumor cells is increased, and its influence on normal cells is reduced. To date, OV has shown its effectiveness and safety in experimental and clinical studies on a variety of tumors. Thus, this review primarily introduces the current status of different genetically engineered OVs used in gene therapy for liver cancer, focuses on the application of OVs and different target genes for current liver cancer therapy, and identifies the problems encountered in OVs-based combination therapy and the corresponding solutions, which will provide new insights into the treatment of liver cancer.

Construction of an expression vector mediated by the dual promoter for prokaryotic and mammalian cell expression system.

The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.

Distance effect characteristic of the matrix attachment region increases recombinant protein expression in Chinese hamster ovary cells.

OBJECTIVES: Previously, we have found that the matrix attachment region (MAR) may confer a 'distance effect' on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism. RESULTS: Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers. CONCLUSIONS: A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system.

[Effects of Different Promoters and MAR Combinations on Transgene Expression of Recombinant CHO Cells].

OBJECTIVE: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells. METHODS: The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of eGFP in CHO cells was analyzed by flow cytometry. The relative copy numbers of eGFP were analyzed by qPCR. RESULTS: Without gMAR expression vector,the expression of eGFP which was driven by CMV-IE promoter was stronger than that of SV40 promoter; gMAR could increase the expression level of eGFP driven by CMV-IE promoter,but did not show any enhancement in SV40 promoter. The expression level of eGFP which containing gMAR on both sides was stronger than that of gMAR on one side driven by CMV-IE promoter; After G418 screening,the expression level of eGFP containing gMAR driven by SV40 promoter wasunstable,the fluorescence gradually weakened,therefore,we only analyzed the expression vector stably expressing the eGFP gene driven by CMV-IE promoter by flow cytometry and qPCR. Compared with the expression vector without gMAR containing CMV-IE promoter,flow cytometry showed that the expression levels of eGFP on one and both sides with gMAR were increased by 9.85-fold and 12.94-fold,respectivley; The result of qPCR showed that the copy number of the eGFP gene without gMAR was set to 1,the copy number of the eGFP gene in the expression vector driven by CMV-IE with gMAR on one side and both sides were 3.68-fold and 9.25-fold,respectively. CONCLUSION: The activity of CMV-IE promoter is stronger than that of SV40 promoter. gMAR can enhance the expression levels of transgene,which may be related to the increase of gene copy number.

The EF-1α promoter maintains high-level transgene expression from episomal vectors in transfected CHO-K1 cells.

In our previous study, we demonstrated that episomal vectors based on the characteristic sequence of matrix attachment regions (MARs) and containing the cytomegalovirus (CMV) promoter allow transgenes to be maintained episomally in Chinese hamster ovary (CHO) cells. However, the transgene expression was unstable and the number of copies was low. In this study, we focused on enhancers, various promoters and promoter variants that could improve the transgene expression stability, expression magnitude (level) and the copy number of a MAR-based episomal vector in CHO-K1 cells. In comparison with the CMV promoter, the eukaryotic translation elongation factor 1 α (EF-1α, gene symbol EEF1A1) promoter increased the transfection efficiency, the transgene expression, the proportion of expression-positive clones and the copy number of the episomal vector in long-term culture. By contrast, no significant positive effects were observed with an enhancer, CMV promoter variants or CAG promoter in the episomal vector in long-term culture. Moreover, the high-expression clones harbouring the EF-1α promoter tended to be more stable in long-term culture, even in the absence of selection pressure. According to these findings, we concluded that the EF-1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number. These results provide valuable information on improvement of transgene stability and the copy number of episomal vectors.

Distance effect of matrix attachment regions on transgene expression in stably transfected Chinese hamster ovary cells.

The ß-globin matrix attachment regions (MARs) were inserted into the 5'-site of the eukaryotic expression vector cassette and DNA fragments 350 and 750 bp in length were inserted into the site to generate expression vectors with varying distances between the expression cassette and MAR. The vectors containing MARs increased chloramphenicol acetyltransferase (CAT) expression levels compared to the negative control vector lacking the MAR; the highest expression increase was 3.8-fold. A greater MAR-transgene distance (750 bp) correlated with a greater increase in transgene expression when compared to the control vector that lacked separation between the MAR and transgene. CAT gene copy numbers were higher in cells transformed with the vector possessing a smaller MAR-transgene distance (350 bp) than in cells belonging to the other three groups. However, MAR-induced transgene expression levels did not exhibit a direct relationship with gene copy number.

Progesterone inhibits the expression of cycloxygenase-2 and interleukin-1ß in neonatal rats with hypoxic ischemic brain damage.

Recent studies found that progesterone (PROG) has a noticeable preventive effect on brain injuries. However, the underlying mechanisms of these effects, particularly on hypoxic ischemic brain damage (HIBD), remain unclear. This study observed the influence of PROG on the expression of cycloxygenase-2 (COX-2) and interleukin-1ß (IL-1ß) in HIBD neonatal rats, and explored the corresponding molecular mechanisms underlying the neuroprotective effect of PROG. Sixty 7-day-old neonatal Wistar rats were divided into sham operation (control), hypoxic ischemia (HI), and drug prophylaxis (PROG) groups, and HIBD animal models were routinely established. The PROG group was intraperitoneally injected with 0.5 g/L PROG at a dosage of 8 mg/kg 30 min before establishment of the model. All the animals were sacrificed 24 h after modeling. Changes in the COX-2 and IL-1ß contents in the brain tissues were determined using enzyme-linked immunosorbent assay. Protein expression was observed using immunohistochemistry, and mRNA expression was determined using reverse transcription polymerase chain reaction. The expression and contents of COX-2 and IL-1ß in the HI group were significantly higher than those in the control group (P < 0.01). Compared with the HI group, the PROG group showed noticeably lower expression and contents of COX-2 and IL-1ß (P < 0.05). In conclusion, PROG can inhibit the expression of COX-2 and IL-1ß in brain tissue, further reducing the level of COX-2 and IL-1ß, which may be one of the mechanisms for protection from HIBD.

Novel and effective screening system for recombinant protein production in CHO cells.

At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.

Patient-derived tumor models: a suitable tool for preclinical studies on esophageal cancer.

Esophageal cancer (EC) is the tenth most common cancer worldwide and has high morbidity and mortality. Its main subtypes include esophageal squamous cell carcinoma and esophageal adenocarcinoma, which are usually diagnosed during their advanced stages. The biological defects and inability of preclinical models to summarize completely the etiology of multiple factors, the complexity of the tumor microenvironment, and the genetic heterogeneity of tumors severely limit the clinical treatment of EC. Patient-derived models of EC not only retain the tissue structure, cell morphology, and differentiation characteristics of the original tumor, they also retain tumor heterogeneity. Therefore, compared with other preclinical models, they can better predict the efficacy of candidate drugs, explore novel biomarkers, combine with clinical trials, and effectively improve patient prognosis. This review discusses the methods and animals used to establish patient-derived models and genetically engineered mouse models, especially patient-derived xenograft models. It also discusses their advantages, applications, and limitations as preclinical experimental research tools to provide an important reference for the precise personalized treatment of EC and improve the prognosis of patients.

Fabrication of Rigid Isocyanate-Based Polyimide Foam Achieved Excellent Use Safety via Synergy between Expandable Graphite and Phosphorus-Containing Polyol.

For the advantages of low cost, excellent thermal insulation, and sound absorption properties, the rigid isocyanate-based polyimide foam (RPIF) presents great application prospects as a building insulation material. However, its inflammability and the accompanying toxic fumes create huge safety hazard. In this paper, reactive phosphate-containing polyol (PPCP) is synthesized and employed with expandable graphite (EG) to obtain RPIF with excellent use safety. EG can be considered as an ideal partner for PPCP to weaken the drawbacks in toxic fume release. Limiting oxygen index (LOI), cone calorimeter test (CCT), and toxic gas results show that the combination of PPCP and EG can synergistically enhance flame retardancy and the use safety of RPIF owing to the unique structure of a dense char layer possessing a flame barrier and toxic gas adsorption effects. When EG and PPCP are simultaneously applied to the RPIF system, the higher EG dosage will bring higher positive synergistic effects in the use safety of RPIF. The most preferred ratio of EG and PPCP is 2:1 (RPIF-10-5) in this study; RPIF-10-5 shows the highest LOI, low CCT results and specific optical density of smoke, and low HCN concentration. This design and the findings are of great significance to improving the application of RPIF.

Multifunctional calcium polyphenol networks reverse the hostile microenvironment of trauma for preventing postoperative peritoneal adhesions.

Abdominal adhesions, a commonly observed complication of abdominal surgery, have a high incidence and adversely affect patients' physical and mental health. The primary causes of abdominal adhesions are intraoperative trauma, acute inflammatory response, bleeding, and foreign body infection. Because most current treatment approaches for abdominal adhesions are limited, improved and novel postoperative anti-adhesion regimens are urgently needed. In this study, we developed calcium polyphenol network (CaPN) microspheres based on the self-assembly of the natural triphenolic compound gallic acid and Ca2+ in solution. The physicochemical properties of CaPNs, including their hemostatic, antibacterial, antioxidant, and anti-inflammatory activities, were investigated in vitro. Bleeding and cecal-abdominal wall adhesion models were established to observe the hemostatic activity of CaPNs and their preventive effect on postoperative abdominal wall adhesion in vivo. The results showed that CaPNs significantly reduced inflammation, oxidative stress, fibrosis, and abdominal adhesion formation and had good hemostatic and antibacterial properties. Our findings suggest a novel strategy for the prevention of postoperative adhesions.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells.

MiR-125b Inhibits Cell Proliferation and Induces Apoptosis in Human Colon Cancer SW480 Cells via Targeting STAT3.

BACKGROUND: Colon cancer is one of the most common types of cancer worldwide. Multiple studies have unveiled the key role of microRNAs (miRNAs) in the development of various types of cancer. However, the mechanism of action of miR-125b in the development and progression of colon cancer remains unknown. OBJECTIVES: In this study, we explored the association of miR-125b and signal transducer and activator of transcription 3 (STAT3) and its role in the proliferation and apoptosis of SW480 colon cancer cells. METHODS: The miR-125b expression in NCM460, SW480, HT29, and HCT8 cells was detected using quantitative real-time polymerase chain reaction (qRT-PCR). SW480 cells were transfected with lentiviruses of GFP-miR-125b and GFP-NC to establish a stable miR-125b overexpression colon cancer cell model and a control model. The targeting relationship between miR-125b and STAT3 was analyzed using bioinformatics and verified by the dual-luciferase reporter gene assay. Cell proliferation and apoptosis were assessed using the Cell Counting Kit-8 assay and TUNEL staining. The expression levels of STAT3, Bcl-2, and Bax were analyzed using Western blot analysis. RESULTS: It was found that the relative mRNA expression of miR-125b was decreased in SW480, HT29, and HCT8 cells compared with that in NCM460 cells (P<0.05). The luciferase reporter gene assay confirmed that miR-125b downregulated the STAT3 gene expression (P<0.05). Overexpression of miR-125b inhibited proliferation and promoted apoptosis in SW480 colon cancer cells and was accompanied by upregulated Bax expression and downregulated Bcl-2 expression (P<0.05). Re-expression of STAT3 promoted cell proliferation and inhibited cell apoptosis, whereas Bcl-2 expression increased, and Bax expression decreased (P<0.05). CONCLUSION: The miR-125b regulates the expression of Bax and Bcl-2 by downregulating the expression of STAT3, thereby inhibiting proliferation and inducing apoptosis of SW480 colon cancer cells.

A trace CH4 detection system based on DAS calibrated WMS technique.

We report the development of a compact near-infrared (NIR) laser-based trace methane (CH4) detection system. This detection system relied on a 2334 nm distributed feedback (DFB) fiber laser as the light source. A parallel dense light-spot pattern multipass gas cell (MGC) with 41.5 m effective absorption path length was utilized to improve the system sensitivity. A self-calibration approach based on direct absorption spectroscopy (DAS) calibrated wavelength modulation spectroscopy (WMS) technique was employed to solve the problem of extra concentration calibration requirement in traditional WMS technique, and to improve the accuracy and stability of the system. According to the Allan deviation analysis, 1-s measurement precision of 0.61 ppmv for DAS and 0.16 ppmv for WMS was obtained, which could be further reduced to 0.11 ppmv for DAS and 0.03 ppmv for WMS by averaging up to 80 s and 50 s, respectively. A week-long continuous atmospheric CH4 concentration measurement was also carried out to demonstrate the long-term performance of our CH4 detection system. With a fast dynamic response characteristics, high-accuracy and high-sensitivity, the proposed detection system is suitable for CH4 measurement in many fields such as atmospheric chemistry analyzation, industrial safety monitoring, agricultural information acquisition, etc.

Screening Strategies for High-Yield Chinese Hamster Ovary Cell Clones.

Chinese hamster ovary (CHO) cells are by far the most commonly used mammalian expression system for recombinant expression of therapeutic proteins in the pharmaceutical industry. The development of high-yield stable cell lines requires processes of transfection, selection, screening and adaptation, among which the screening process requires tremendous time and determines the level of forming highly productive monoclonal cell lines. Therefore, how to achieve productive cell lines is a major question prior to industrial manufacturing. Cell line development (CLD) is one of the most critical steps in the production of recombinant therapeutic proteins. Generation of high-yield cell clones is mainly based on the time-consuming, laborious process of selection and screening. With the increase in recombinant therapeutic proteins expressed by CHO cells, CLD has become a major bottleneck in obtaining cell lines for manufacturing. The basic principles for CLD include preliminary screening for high-yield cell pool, single-cell isolation and improvement of productivity, clonality and stability. With the development of modern analysis and testing technologies, various screening methods have been used for CLD to enhance the selection efficiency of high-yield clonal cells. This review provides a comprehensive overview on preliminary screening methods for high-yield cell pool based on drug selective pressure. Moreover, we focus on high throughput methods for isolating high-yield cell clones and increasing the productivity and stability, as well as new screening strategies used for the biopharmaceutical industry.

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells.

Recombinant antibodies are rapidly developing therapeutic agents; approximately 40 novel antibody molecules enter clinical trials each year, most of which are produced from Chinese hamster ovary (CHO) cells. However, one of the major bottlenecks restricting the development of antibody drugs is how to perform high-level expression and production of recombinant antibodies. The high-efficiency expression and quality of recombinant antibodies in CHO cells is determined by multiple factors. This review provides a comprehensive overview of several state-of-the-art approaches, such as optimization of gene sequence of antibody, construction and optimization of high-efficiency expression vector, using antibody expression system, transformation of host cell lines, and glycosylation modification. Finally, the authors discuss the potential of large-scale production of recombinant antibodies and development of culture processes for biopharmaceutical manufacturing in the future.

Murine RANK protein's inhibition of bone resorption.

OBJECTIVE: The objective was to study the inhibitory effects of recombinant murine receptor activator of nuclear factor κB (RANK) protein on osteoclasts in vivo and in vitro. METHODS: The RANK protein was added to the cocultures of osteoclasts at concentrations of 10(-6), 10(-5), and 10(-4) g/L. The morphology and number of osteoclasts were examined. Female KM mice were ovariectomized and treated with RANK protein at 5 mg/kg body weight. Biochemical markers of bone metabolism, bone mineral density, and bone morphology were examined. RESULTS: Three days after RANK treatment, the numbers of tartrate-resistant acid phosphatase-positive osteoclasts and resorption pits in bone slices decreased significantly in each treatment group, with the most significant decrease observed in the 10(-4) g/L group. Compared with the control group in vivo, the RANK-treated group exhibited higher bone mineral density and nearly complete inhibition of tartrate-resistant acid phosphatase-positive osteoclasts in bone slices. CONCLUSIONS: Recombinant murine RANK protein effectively inhibits the activity of osteoclasts and the resulting bone resorption.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying IL-24/endostatin enhances glioma therapy.

Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.

Classification of soybean frogeye leaf spot disease using leaf hyperspectral reflectance.

In this study, the feasibility of classifying soybean frogeye leaf spot (FLS) is investigated. Leaf images and hyperspectral reflectance data of healthy and FLS diseased soybean leaves were acquired. First, image processing was used to classify FLS to create a reference for subsequent analysis of hyperspectral data. Then, dimensionality reduction methods of hyperspectral data were used to obtain the relevant information pertaining to FLS. Three single methods, namely spectral index (SI), principal component analysis (PCA), and competitive adaptive reweighted sampling (CARS), along with a PCA and SI combined method, were included. PCA was used to select the effective principal components (PCs), and evaluate SIs. Characteristic wavelengths (CWs) were selected using CARS. Finally, the full wavelengths, CWs, effective PCs, SIs, and significant SIs were divided into 14 datasets (DS1-DS14) and used as inputs to build the classification models. Models' performances were evaluated based on the classification accuracy for both the overall and individual classes. Our results suggest that the FLS comprised of five classes based on the proportion of total leaf surface covered with FLS. In the PCA and SI combination model, 5 PCs and 20 SIs with higher weight coefficient of each PC were extracted. For hyperspectral data, 20 CWs and 26 effective PCs were also selected. Out of the 14 datasets, the model input variables provided by five datasets (DS2, DS3, DS4, DS10, and DS11) were more superior than those of full wavelengths (DS1) both in support vector machine (SVM) and least squares support vector machine (LS-SVM) classifiers. The models developed using these five datasets achieved overall accuracies ranging from 91.8% to 94.5% in SVM, and 94.5% to 97.3% in LS-SVM. In addition, they improved the classification accuracies by 0.9% to 3.6% (SVM) and 0.9% to 3.7% (LS-SVM).