Determination of aflatoxins in rice from Penang, Malaysia by dispersive liquid-liquid micro-extraction and LC-MS/MS.

Rice is one of the crops cultivated in Malaysia, and it is the main diet for most of the population. In this study, dispersive liquid-liquid micro-extraction (DLLME) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to develop, optimise and validate a reliable, easy-to-use and quick approach to detect aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). The extraction recoveries in DLLME were enhanced by the addition of 5% salt, utilising chloroform as the extraction solvent and acetonitrile as the dispersive solvent. The DLLME parameters - the extraction solvent volume, salt concentration and dispersive solvent volume were optimised with Box-Behnken design (BBD) and response surface methodology (RSM). Under optimised experimental conditions, excellent linearity was obtained with a limit of detection (LOD) ranging from 0.125 to 0.25 ng g-1, a limit of quantitation (LOQ) ranging from 0.25 to 0.3 ng g-1 and a correlation value (R2) of 0.990. The matrix effects were between -11.1% and 19.9%, and recoveries ranged from 87.4% to 117.3%. The optimised and validated method was used effectively to assess aflatoxins contamination in 20 commercial rice samples collected from local supermarkets in Penang, Malaysia. AFB1 was detected at 0.41-0.43 ng g-1 in two rice samples, below the regulatory limit.

Detection of dynorphin 1-17 biotransformation fragments in human nasal polyps by UPLC-QTOF-MS.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent inflammation of the sinonasal mucosa. CRSwNP treatments are associated with inconsistent efficacy and recurrence of symptoms. Dynorphin 1-17 (DYN 1-17) and its fragments have been shown to modulate the immune response in various inflammatory conditions. This study aimed to investigate the effect of different pH and degrees of inflammation on DYN 1-17 metabolism in human CRSwNP tissues. DYN 1-17 was incubated with grade 3 and grade 4 inflamed tissues of CRSwNP patients at pH 5.5 and pH 7.4 over a range of incubation periods. The resulting fragments were identified using an ultra-performance liquid chromatography (UPLC) system coupled to quadrupole-time of flight (QTOF) mass spectrometry based on their accurate mass. The rate of DYN 1-17 fragmentation was slower at pH 5.5 in comparison to pH 7.4. The extent and rate of metabolism of DYN 1-17 were much lower in grade 3 inflamed tissue (31-32 fragments) than in grade 4 (34-41 fragments). N-Terminal fragments (DYN 1-15, 1-11, 1-10, and 1-6) were metabolized slower at pH 5.5 as compared to pH 7.4. DYN 1-12, 1-8, 2-10, 4-10, 5-10, and 8-14 were only observed under the inflammatory pH while DYN 5-17 and 6-17 were only identified upon incubation with grade 4 CRSwNP tissues. DYN 1-17 metabolism was significantly affected by the pH level and the severity of the inflammation of CRSwNP tissues, indicating the potential roles of DYN 1-17 and its fragments in modulating the inflammatory response and their avenue as therapeutics in future studies.

Glycoproteomics-based liquid biopsy: translational outlook for colorectal cancer clinical management in Southeast Asia.

Colorectal cancer (CRC) signifies a significant healthcare challenge in Southeast Asia. Despite advancements in screening approaches and treatment modalities, significant medical gaps remain, ranging from prevention and early diagnosis to determining targeted therapy and establishing personalized approaches to managing CRC. There is a need to expand more validated biomarkers in clinical practice. An advanced technique incorporating high-throughput mass spectrometry as a liquid biopsy to unravel a repertoire of glycoproteins and glycans would potentially drive the development of clinical tools for CRC screening, diagnosis and monitoring, and it can be further adapted to the existing standard-of-care procedure. Therefore this review offers a perspective on glycoproteomics-driven liquid biopsy and its potential integration into the clinical care of CRC in the southeast Asia region.

An Empirical Analysis of Sacha Inchi (Plantae: Plukenetia volubilis L.) Seed Proteins and Their Applications in the Food and Biopharmaceutical Industries.

Sacha Inchi (Plukenetia volubilis L.) is a plant native in the Amazon rainforest in South America known for its edible seeds, which are rich in lipids, proteins, vitamin E, polyphenols, minerals, and amino acids. Rural communities in developing nations have been using this plant for its health benefits, including as a topical cream for rejuvenating and revitalising skin and as a treatment for muscle pain and rheumatism. Although Sacha Inchi oil has been applied topically to soften skin, treat skin diseases, and heal wounds, its protein-rich seeds have not yet received proper attention for extensive investigation. Proteins in Sacha Inchi seeds are generally known to have antioxidant and antifungal activities and are extensively used nowadays in making protein-rich food alternatives worldwide. Notably, large-scale use of seed proteins has begun in nanoparticle and biofusion technologies related to the human health-benefitting sector. To extract and identify their proteins, the current study examined Sacha Inchi seeds collected from the Malaysian state of Kedah. Our analysis revealed a protein concentration of 73.8 ± 0.002 mg/g of freeze-dried seed flour. Employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and PEAKS studio analysis, we identified 217 proteins in the seed extract, including 152 with known proteins and 65 unknown proteins. This study marks a significant step towards comprehensively investigating the protein composition of Sacha Inchi seeds and elucidating their potential applications in the food and biopharmaceutical sectors. Our discoveries not only enhance our knowledge of Sacha Inchi's nutritional characteristics but also pave the way for prospective research and innovative advancements in the realms of functional food and health-related domains.

Protein Profiling in Human Papillomavirus-Associated Cervical Carcinogenesis: Cornulin as a Biomarker for Disease Progression.

Nearly 90% of cervical cancers are linked to human papillomavirus (HPV). Uncovering the protein signatures in each histological phase of cervical oncogenesis provides a path to biomarker discovery. The proteomes extracted from formalin-fixed paraffin-embedded tissues of the normal cervix, HPV16/18-associated squamous intraepithelial lesion (SIL), and squamous cell carcinoma (SCC) were compared using liquid chromatography-mass spectrometry (LC-MS). A total of 3597 proteins were identified, with 589, 550, and 1570 proteins unique to the normal cervix, SIL, and SCC groups, respectively, while 332 proteins overlapped between the three groups. In the transition from normal cervix to SIL, all 39 differentially expressed proteins were downregulated, while all 51 proteins discovered were upregulated in SIL to SCC. The binding process was the top molecular function, while chromatin silencing in the SIL vs. normal group, and nucleosome assembly in SCC vs. SIL groups was the top biological process. The PI3 kinase pathway appears crucial in initiating neoplastic transformation, while viral carcinogenesis and necroptosis are important for cell proliferation, migration, and metastasis in cervical cancer development. Annexin A2 and cornulin were selected for validation based on LC-MS results. The former was downregulated in the SIL vs. normal cervix and upregulated in the progression from SIL to SCC. In contrast, cornulin exhibited the highest expression in the normal cervix and lowest in SCC. Although other proteins, such as histones, collagen, and vimentin, were differentially expressed, their ubiquitous expression in most cells precluded further analysis. Immunohistochemical analysis of tissue microarrays found no significant difference in Annexin A2 expression between the groups. Conversely, cornulin exhibited the strongest expression in the normal cervix and lowest in SCC, supporting its role as a tumor suppressor and potential biomarker for disease progression.

Label-free quantitative mass spectrometry analysis of the circadian proteome of Drosophila melanogaster lethal giant larvae mutants reveals potential therapeutic effects of melatonin.

Mutation in the Drosophila melanogaster lethal giant larvae (lgl), a tumor suppressor gene with a well-established role in cellular polarity, is known to results in massive cellular proliferation and neoplastic outgrowths. Although the tumorigenic properties of lgl mutant have been previously studied, however, little is known about its consequences on the proteome. In this study, mass spectrometry-based label-free quantitative proteomics was employed to investigate the changes in the head and intestinal tissues proteins of Drosophila melanogaster, due to lgl mutation and following treatment with melatonin. Additionally, to uncover the time-influenced variations in the proteome during tumorigenesis and melatonin treatment, the rhythmic expression of proteins was also investigated at 6-h intervals within 24-h clock. Together, the present study has identified 434 proteins of altered expressions (p < 0.05 and fold change ±1.5) in the tissues of flies in response to lgl mutation as well as posttreatment with melatonin. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed proteins revealed that lgl mutation had significantly affected the biological functions, including metabolism, and protein synthesis and degradation, in flies' tissues. Besides, melatonin had beneficially mitigated the deleterious effects of lgl mutation by reversing the alterations in protein expression closer to baseline levels. Further, changes in protein expression in the tissues due to lgl mutation and melatonin treatment were found rhythmically orchestrated. Together, these findings provide novel insight into the pathways involved in lgl-induced tumorigenesis as well as demonstrated the efficacy of melatonin as a potential anticancer agent. Data are available via ProteomeXchange with identifier PXD033191.

Phosphorylation of interferon regulatory factor 9 (IRF9).

BACKGROUND: IRF9 is a transcription factor that mediates the expression of interferon-stimulated genes (ISGs) through the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) pathway. The JAK-STAT pathway is regulated through phosphorylation reactions, in which all components of the pathway are known to be phosphorylated except IRF9. The enigma surrounding IRF9 regulation by a phosphorylation event is intriguing. As IRF9 plays a major role in establishing an antiviral state in host cells, the topic of IRF9 regulation warrants deeper investigation. METHODS: Initially, total lysates of 2fTGH and U2A cells (transfected with recombinant IRF9) were filter-selected and concentrated using phosphoprotein enrichment assay. The phosphoprotein state of IRF9 was further confirmed using Phos-tag™ assay. All protein expression was determined using Western blotting. Tandem mass spectrometry was conducted on immunoprecipitated IRF9 to identify the phosphorylated amino acids. Finally, site-directed mutagenesis was performed and the effects of mutated IRF9 on relevant ISGs (i.e., USP18 and Mx1) was evaluated using qPCR. RESULTS: IRF9 is phosphorylated at S252 and S253 under IFNß-induced condition and R242 under non-induced condition. Site-directed mutagenesis of S252 and S253 to either alanine or aspartic acid has a modest effect on the upregulation of USP18 gene-a negative regulator of type I interferon (IFN) response-but not Mx1 gene. CONCLUSION: Our preliminary study shows that IRF9 is phosphorylated and possibly regulates USP18 gene expression. However, further in vivo studies are needed to determine the significance of IRF9 phosphorylation.

Factors influencing COVID-19 vaccination intention among university students: A cross-sectional study in Malaysia.

Vaccination is crucial in controlling the spread of the coronavirus disease 2019 (COVID-19) that triggered the pandemic, but herd immunity can only work with high vaccination coverage in the population. This study aims to measure the COVID-19 knowledge level and determine the factors influencing COVID-19 vaccination intention among university students in Malaysia. A cross-sectional online survey was carried out with 1,274 Malaysian university students in July 2021. Univariate and multivariate analyses were employed to examine the relationships between the study variables. Results showed that the majority of university students had an acceptable level of knowledge of COVID-19. The knowledge, risk perception of COVID-19, social norms, and perceived benefit of COVID-19 vaccination were positively associated with vaccination intention. However, perceived trust in information sources of COVID-19 vaccination and the government's response to COVID-19 did not affect the university students' desire to receive the vaccination. These findings are essential for health policymakers and healthcare providers to implement evidence-based interventions to increase COVID-19 vaccination uptake among university students.

Proteomic analysis of Malaysian Horseshoe crab (Tachypleus gigas) hemocytes gives insights into its innate immunity host defence system and other biological processes.

Horseshoe crabs are one of the most studied invertebrates due to their remarkable innate immunity mechanism and biological processes. In this work, the proteins of the lipopolysaccharides (LPS)-stimulated and non-stimulated hemocytes of Malaysian Tachypleus gigas were profiled using LC-MS/MS. A total of 154 proteins were identified in both types of samples. Additionally, seventy-seven proteins were commonly found in both conditions, while 52 and 25 proteins were uniquely found in the LPS-stimulated and non-stimulated hemocytes, respectively. ATP-dependent energy-generating proteins such as actins and BLTX actin-related proteins were detected in both stimulated and non-stimulated T. gigas hemocytes, but more of such proteins were found in the former type. Proteins such as tachylectin-2, coagulogen, c-reactive proteins, histones, hemocyanin, and DNA polymerase, which play key roles in the organism's innate immunity, were differentially expressed in the hemocytes following LPS challenge. In conclusion, the proteins identified in the hemolymph of T. gigas are vital for the organism's molecular functions, biological processes, and activation of innate immunity.

Recent insights on gene expression studies on Hevea Brasiliensis fatal leaf fall diseases.

Hevea brasiliensis is one of the most important agricultural commodities globally, heavily cultivated in Southeast Asia. Fatal leaf fall diseases cause aggressive leaf defoliation, linked to lower latex yield and death of crops before maturity. Due to the significant consequences of the disease to H. brasiliensis, the recent gene expression studies from four fall leaf diseases of H. brasiliensis were gathered; South American leaf blight, powdery mildew, Corynespora cassiicola and Phytophthora leaf fall disease. The differential analysis observed the pattern of commonly expressed genes upon fungi triggers using RT-PCR, DDRT-PCR, Real-time qRT-PCR and RNA-Seq. We have observed that RNA-Seq is the best tool to seek novel genes. Among the identified genes with defence-against fungi were pathogenesis-related genes such as ß-1,3-glucanase and chitinase, the reactive oxygen species, and the phytoalexin biosynthesis. This manuscript also provided functional elaboration on the responsive genes and predicted possible biosynthetic pathways to identify and characterise novel genes in the future. At the end of the manuscript, the PCR methods and proteomic approaches were presented for future molecular and biochemical studies in the related diseases to H. brasiliensis.

Extraction and identification of biologically important proteins from the medicinal plant God's crown (Phaleria macrocarpa).

The fruit and leaf of God's crown (Phaleria macrocarpa) have been traditionally used to treat a wide variety of diseases. However, the proteins of this tropical plant are still heavily understudied. Three protein extraction methods; phenol (Phe), trichloroacetic acid (TCA)-acetone-phenol (TCA-A-Phe), and ultrasonic (Ult) were compared on the fruit and leaf of P. macrocarpa. The Phe extraction method showed the highest percentage of recovered protein after the resolubilization process for both leaf (12.24%) and fruit (30.41%) based on protein yields of the leaf (6.15 mg/g) and fruit (36.98 mg/g). Phe and TCA-A-Phe extraction methods gave well-resolved bands over a wide range of molecular weights through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following liquid chromatography-tandem mass spectrometry analysis, proteins identified through the Phe extraction method were 30%-35% enzymatic proteins, including oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases that possess various biological functions. PRACTICAL APPLICATIONS: Every part of God's crown plant is traditionally consumed to treat various illnesses. While plant's benefits are well known and have led to a plethora of health products, the proteome remains mostly unknown. This study compares three protein extraction methods for the leaf and fruit of P. macrocarpa and identifies their proteins thru LC-MS/MS coupled with PEAKS. These method comparisons can be a guide for works on other plants as well. In addition, the proteomics data from this study may shed light on the functional properties of these plant parts and their products.

Protein Composition and Biomedical Potential of the Skin Secretion of Hylarana erythraea (Schlegel, 1837) (Anura: Ranidae) from Langkawi Archipelago, Kedah, Peninsular Malaysia.

The skin secretion of amphibians is known for its high content of bioactive compounds. These bioactive compounds are essential for the advancement of biomedical industries. Four wild green paddy frogs, Hylarana erythraea, were collected from various habitat types within the Langkawi Archipelago. These frogs' skin secretions were collected, extracted, and analysed for their protein compounds together with biomedical potentials using liquid chromatography-mass spectrometry (LC-MS). The total protein concentration of H. erythraea skin secretions was determined as 0.269 mg/mL. Based on the UniProt (Anura) database, we identified 29 proteins. These proteins were categorised as antimicrobial (AMP) (38%), followed by hormone (17%), enzyme (17%), unreviewed proteins (17%), structural proteins (7%), and regulatory proteins (4%). The AMPs identified were from the family of esculentin-1, esculentin-2, brevinin-1, and frenatin-4, while the hormones belonged to the cholecystokinin group. The enzymes detected were adenylate cyclase 9, the suppressor of tumorigenicity 14 protein homolog, and the HGF activator. The structural proteins belonged to toe pad keratin 2 and Krt5.7 proteins, while the single regulatory protein is CCR4-NOT transcription complex subunit 6-like. These proteins have a wide range of biomedical importance, such as wound healings, facilitate digestions, anti-tumours, and anti-cancer effect.

Dispersive Liquid-Liquid Microextraction (DLLME) and LC-MS/MS Analysis for Multi-Mycotoxin in Rice Bran: Method Development, Optimization and Validation.

Rice bran, a by-product of the rice milling process, has emerged as a functional food and being used in formulation of healthy food and drinks. However, rice bran is often contaminated with numerous mycotoxins. In this study, a method to simultaneous detection of aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), deoxynivalenol (DON), fumonisins (FB1 and FB2), sterigmatocystin (STG), T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and zearalenone (ZEA) in rice bran was developed, optimized and validated using dispersive liquid-liquid microextraction (DLLME) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In DLLME, using a solvent mixture of methanol/water (80:20, v/v) as the dispersive solvent and chloroform as the extraction solvent with the addition of 5% salt improved the extraction recoveries (63-120%). The developed method was further optimized using the response surface methodology (RSM) combined with Box-Behnken Design (BBD). Under the optimized experimental conditions, good linearity was obtained with a correlation coefficient (r2) ≥ 0.990 and a limit of detection (LOD) between 0.5 to 50 ng g-1. The recoveries ranged from 70.2% to 99.4% with an RSD below 1.28%. The proposed method was successfully applied to analyze multi-mycotoxin in 24 rice bran samples.

Synergistic Roles of Curcumin in Sensitising the Cisplatin Effect on a Cancer Stem Cell-Like Population Derived from Non-Small Cell Lung Cancer Cell Lines.

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.

The effects of high-fat diet and metformin on urinary metabolites in diabetes and prediabetes rat models.

High-fat diet (HFD) interferes with the dietary plan of patients with type 2 diabetes mellitus (T2DM). However, many diabetes patients consume food with higher fat content for a better taste bud experience. In this study, we examined the effect of HFD on rats at the early onset of diabetes and prediabetes by supplementing their feed with palm olein oil to provide a fat content representing 39% of total calorie intake. Urinary profile generated from liquid chromatography-mass spectrometry analysis was used to construct the orthogonal partial least squares discriminant analysis (OPLS-DA) score plots. The data provide insights into the physiological state of an organism. Healthy rats fed with normal chow (NC) and HFD cannot be distinguished by their urinary metabolite profiles, whereas diabetic and prediabetic rats showed a clear separation in OPLS-DA profile between the two diets, indicating a change in their physiological state. Metformin treatment altered the metabolomics profiles of diabetic rats and lowered their blood sugar levels. For prediabetic rats, metformin treatment on both NC- and HFD-fed rats not only reduced their blood sugar levels to normal but also altered the urinary metabolite profile to be more like healthy rats. The use of metformin is therefore beneficial at the prediabetes stage.

Hevea brasiliensis latex proteomics: a review of analytical methods and the way forward.

Natural rubber or latex from the Hevea brasiliensis is an important commodity in various economic sectors in today's modern society. Proteins have been detected in latex since the early twentieth century, and they are known to regulate various biological pathways within the H. brasiliensis trees such as the natural rubber biosynthesis, defence against pathogens, wound healing, and stress tolerance. However, the exact mechanisms of the pathways are still not clear. Proteomic analyses on latex have found various proteins and revealed how they fit into the mechanisms of the biological pathways. In the past three decades, there has been rapid latex protein identification due to the improvement of latex protein extraction methods, as well as the emergence of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). In this manuscript, we reviewed the methods of latex protein extraction that keeps on improving over the past three decades as well as the results of numerous latex protein identification and quantitation.

Comparative proteomic analysis of different stages of breast cancer tissues using ultra high performance liquid chromatography tandem mass spectrometer.

BACKGROUND: Breast cancer is the fifth most prevalent cause of death among women worldwide. It is also one of the most common types of cancer among Malaysian women. This study aimed to characterize and differentiate the proteomics profiles of different stages of breast cancer and its matched adjacent normal tissues in Malaysian breast cancer patients. Also, this study aimed to construct a pertinent protein pathway involved in each stage of cancer. METHODS: In total, 80 samples of tumor and matched adjacent normal tissues were collected from breast cancer patients at Seberang Jaya Hospital (SJH) and Kepala Batas Hospital (KBH), both in Penang, Malaysia. The protein expression profiles of breast cancer and normal tissues were mapped by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Gel-Eluted Liquid Fractionation Entrapment Electrophoresis (GELFREE) Technology System was used for the separation and fractionation of extracted proteins, which also were analyzed to maximize protein detection. The protein fractions were then analyzed by tandem mass spectrometry (LC-MS/MS) analysis using LC/MS LTQ-Orbitrap Fusion and Elite. This study identified the proteins contained within the tissue samples using de novo sequencing and database matching via PEAKS software. We performed two different pathway analyses, DAVID and STRING, in the sets of proteins from stage 2 and stage 3 breast cancer samples. The lists of molecules were generated by the REACTOME-FI plugin, part of the CYTOSCAPE tool, and linker nodes were added in order to generate a connected network. Then, pathway enrichment was obtained, and a graphical model was created to depict the participation of the input proteins as well as the linker nodes. RESULTS: This study identified 12 proteins that were detected in stage 2 tumor tissues, and 17 proteins that were detected in stage 3 tumor tissues, related to their normal counterparts. It also identified some proteins that were present in stage 2 but not stage 3 and vice versa. Based on these results, this study clarified unique proteins pathways involved in carcinogenesis within stage 2 and stage 3 breast cancers. CONCLUSIONS: This study provided some useful insights about the proteins associated with breast cancer carcinogenesis and could establish an important foundation for future cancer-related discoveries using differential proteomics profiling. Beyond protein identification, this study considered the interaction, function, network, signaling pathway, and protein pathway involved in each profile. These results suggest that knowledge of protein expression, especially in stage 2 and stage 3 breast cancer, can provide important clues that may enable the discovery of novel biomarkers in carcinogenesis.

Bioactive Proteins in Channa striata Promote Wound Healing through Angiogenesis and Cell Proliferation.

BACKGROUND: Channa striata are speculated to contain bioactive proteins with the ability to enhancing wound healing. It is commonly consumed after surgery for a faster recovery of the wound. OBJECTIVE: To identify the bioactive proteins and evaluate their ability in cell proliferation and angiogenesis promotion. MATERIAL AND METHODS: Freeze-Dried Water Extracts (FDWE) and Spray-Dried Water Extracts (SDWE) of C. striata were tested with MTT assay using EA.hy926 endothelial cell line and ex-vivo aortic ring assay. Later the proteins were fractionated and analysed using an LC-QTOF mass spectrometer. The data generated were matched with human gene database for protein similarity and pathway identification. RESULTS: Both samples have shown positive cell proliferation and pro-angiogenic activity. Four essential proteins/genes were identified, which are collagen type XI, actin 1, myosin light chain and myosin heavy chain. The pathways discovered that related to these proteins are integrin pathway, Slit-Robo signalling pathway and immune response C-C Chemokine Receptor-3 signalling pathway in eosinophils, which contribute towards wound healing mechanism. CONCLUSIONS: The results presented have demonstrated that C. striata FDWE and SDWE protein fractions contain bioactive proteins that are highly similar to human proteins and thus could be involved in the wound healing process via specific biological pathways.

Development of a rapid and improved two-dimensional electrophoresis method for the separation of protein lysate.

Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.