Malaysian Applied Biology

DAMD And ISSR DNA Molecular Analyses of Regenerated Cryopreserved Dendrobium Sabin Blue’s Protocorm-Like Bodies (PLBS)

Jessica Jeyanthi James Antony — 2025-09-30

The Orchidaceae is known as one of the most species-rich families of the plant kingdom. Orchids are generally declining in their natural habitat due to habitat loss and overharvesting for ornamental and medicinal purposes. Dendrobium Sabin Blue is widely grown as a cut flower and pot plant and is also popular for its deep violet-blue flowers. Developing these new orchid hybrids is tedious work; it is important to preserve them. Tissue culture and cryopreservation of plants can be employed to produce plantlets on an industrial scale. However, it may lead to genetic instability due to somaclonal variation. This study is to assess the genetic stability of regenerated cryopreserved and non-cryopreserved PLBs in comparison to stock culture PLBs using directed amplification of minisatellite DNA regions (DAMD) and inter-simple sequence repeat (ISSR) DNA molecular analyses. In general, regenerated explants should be identical to the mother plant. Seventeen (17) DAMD primers and twenty (20) ISSR primers were used to assess genetic stability between the 4-week-old cryopreserved/non-cryopreserved PLBs and the PLBs of the parent culture. Finally, DAMD and ISSR analyses confirmed the occurrence of 7% polymorphism and monomorphism, respectively, in the regenerated cryopreserved PLBs. Future studies should include further evaluation of somaclonal variations for long-term maintenance of cryopreserved PLBs.

Differentially Expressed Proteins in Corynebacterium pseudotuberculosis During Biofilm Formation

Anati Abd Rashid Syaida — 2025-09-30

Corynebacterium pseudotuberculosis is a non-motile, β-hemolytic bacterium and causative factor of caseous lymphadenitis. The disease affects sheep and goats, causing impaired wool production, weight loss, and carcass condemnation. Our previous work has elucidated the morphology, heterogeneity, and antimicrobial susceptibility of C. pseudotuberculosis biofilm. However, the information on proteome expression underlying C. pseudotuberculosis biofilm development remains scarce. Thus, the objective of the present work is to compare the whole-cell proteome profiles between planktonic and biofilm fractions of C. pseudotuberculosis and identify C. pseudotuberculosis proteins and biological pathways showing differential expression. C. pseudotuberculosis biofilm was grown in a six-well microplate for 24 hr at 37°C. Polyacrylamide gel electrophoresis combined with tandem mass spectrometry and bioinformatics analysis was conducted to analyze proteome expression. Results demonstrated differential expression of seven SDS-PAGE protein bands (33.7 – 150 kDa) in comparison between the planktonic and biofilm fractions of C. pseudotuberculosis. Overall, 711 proteins that showed differential expression were successfully identified, while the protein-protein interaction network revealed a total of 3868 functional linkages among the differentially expressed proteins. Fifty-seven hub proteins with more than 10 functional linkages were identified, including large subunit ribosomal protein L3, translation initiation factor IF-2, multifunctional oxoglutarate decarboxylase, and DNA-dependent RNA polymerase. Functional enrichment analysis revealed the association of differentially expressed C. pseudotuberculosis proteins with secondary metabolite metabolism (p-value<0.05). In conclusion, differential protein expressions in C. pseudotuberculosis may modulate adaptive responses to environmental stressors, thereby promoting biofilm formation.

In Vitro Evaluation of α-Amylase, α-Glucosidase Inhibition Activity and Protein Denaturation Inhibition of Polysaccharides from Leaves and Seeds of Zygophyllum album L.

Malika Seddiki — 2025-09-30

The present study investigates anti-hyperglycemic activity and protein denaturation inhibition of polysaccharide extracts from leaves (ZF) and seeds (ZG) of Zygophyllum album L (Zygophyllaceae), a wild plant harvested in the south-east of the Algerian Sahara. The global composition of the extracts was determined by spectrophotometric assays, and the monosaccharide composition was determined by TLC and HPLC-RID. The extraction yields were 0.53 ± 0.04% and 2.33 ± 0.52% for ZF and ZG, respectively. The leaf extract is rich in uronic acids (32.91 ± 0.23%) compared to the seed extract (5.98 ± 1.31%). The analysis of both extracts indicates that their composition of constituent monosaccharides is similar. ZF consists of 20.90% arabinose, 24.16% galactose, 22.88% xylose, 14.71% rhamnose, and 17.34% glucose, and ZG of 21.64% arabinose, 31.76% galactose, 16.95% xylose, 17.25% rhamnose, and 12.39% glucose. Their FT-IR spectra display the characteristic bands of polysaccharides. Seed extract shows a significant inhibitory effect against protein denaturation (IC₅₀ of 0.93 ± 0.02 mg/mL & 22.75 ± 1.41 mg/mL for ZG & ZF, respectively). However, the leaf extract, with a low α-amylase inhibitory activity (IC₅₀ of 90.16 ± 5.38 mg/mL) and a high α-glucosidase inhibitory power (IC₅₀ of 5.44 ± 0.50 mg/mL), seems to be effective in reducing postprandial hyperglycemia.

Enhanced Cell Harvesting for Lipid Production by Lipomyces maratuensis InaCC Y720 Using Flocculation Induction

Theresia Kusumawati — 2025-09-30

Lipomyces maratuensis InaCC Y720 is a yeast from Indonesia that is known to be able to produce lipids on a production medium that has been optimized in the previous study. One of the efforts to increase the harvesting of yeast cells needed on a production scale larger than the laboratory scale can be done by flocculation induction. This study showed that flocculation induction significantly increased the cell harvest of Lipomyces maratuensis InaCC Y720, with the biomass obtained increasing up to 2.7 times compared to the method without flocculation induction. The combination of pH 9 adjustment and the addition of Ca²⁺ ions proved to be the optimal conditions to induce flocculation, resulting in cell aggregations that appeared adherent based on scanning electron microscopy (SEM). These findings suggest that the flocculation induction not only improves cell harvesting efficiency but also has the potential to be applied in industrial-scale lipid production as a more effective harvesting method. Although the main composition of the fatty acids produced remained consistent, there were differences in the percentage of each fatty acid between harvesting methods with and without flocculation induction. However, to maximize the utilization of lipids from flocculated cells, further research is needed to develop more efficient extraction methods. Thus, this study provides a solid foundation for further exploration in the optimization of lipid production using oleaginous yeast.

Regulation of OsSAP8 Promoter in Response to Abiotic Stresses

Sitti ‘Aisyah Mohd Roszelin — 2025-09-30

Abiotic stresses such as drought, salinity and extreme temperatures pose significant challenges to crop production, particularly impacting rice yield and quality. These stresses are exacerbated by climate change and the escalation of the human population. Plant adaptation to abiotic stresses involves intricate molecular mechanisms, including gene expression alterations, metabolic adjustments, and stress-responsive gene activation. Phytohormones play pivotal roles in regulating these adaptive responses, by playing a central role in regulating plant growth and enhancing resilience to stress. Previous studies have shown that Oryza sativa Stress-associated protein 8 (OsSAP8) enhanced plant tolerance to drought and salinity stresses throughout the growth and developmental stages. In this study, we focused on the OsSAP8 promoter, especially the phytohormone-responsive Cis-Regulatory Elements (CREs), to deepen our understanding of its regulation under abiotic stress conditions. Promoter analysis identified several CREs associated with Abscisic Acid (ABA), Gibberellic Acid (GA) and Methyl-Jasmonate (MeJA) phytohormones. Subsequently, promoter deletion was performed using two different lengths of OsSAP8 promoter fragments, comprising different sets of phytohormone CREs. Promoter-β-glucuronidase (GUS) fusion constructs in transgenic Arabidopsis plants revealed that the truncated promoter fragment of pOsSAP8(934pb)::GUS exhibited stronger GUS activity compared to the full-length promoter, pOsSAP8(1801pb)::GUS under drought and salinity stresses. This suggests that the CREs responsible for OsSAP8 expression under stress conditions are located within this shorter promoter region. These findings underscore the importance of OsSAP8 in plant stress responses and provide a foundation for future research on enhancing agricultural sustainability amid changing environmental conditions.

Shorea macrophylla (Engkabang) Endosperm Extract Elevates Lipogenesis and Adipogenesis in 3T3-L1 Mouse Adipocyte Cells

Hung Hui Chung — 2025-09-30

The Shorea macrophylla endosperm, also known as illipe nuts, has a high lipid content, making it a favoured dietary choice for wild animals. Its composition is an interesting aspect to investigate, as it is still unknown to date despite its superior high-quality lipid profile. In this research, we conducted endosperm extraction using various solvents to investigate potential lipogenesis-inducing compounds. The acetone showed the highest endosperm extraction efficiency at 38.30% while other solvents achieved efficiency above 30%, except for ethanol and methanol. GCMS analysis revealed common compounds across extracts, with methyl stearate and methyl elaidate being among the most prevalent, except in acetone extraction. Molecular docking experiments demonstrated good binding scores between these compounds and lipogenesis-related proteins, particularly highlighting methyl elaidate's potential. In vitro studies conducted on 3T3-L1 cells confirmed an increase in triglyceride levels with methanol and diethyl ether extracts. Both experimental and computational analyses strengthen the potential of methyl elaidate as a pivotal lipogenesis inducer, with potential implications for enhancing fat storage in meat quality when incorporated into vertebrate diets. The effective lipogenesis-inducing elements mentioned can then be incorporated in the diet of livestock with the hope of increasing the amount of fat stored in meat and improving its overall quality.

Pleurotus pulmonarius Stalk Waste Extract Supplementation Modulates Serum Proteins Level in Red Hybrid Tilapia Oreochromis sp. upon PAMPs Challenge

Joo Jie Ching — 2025-09-30

Utilization of functional feeds to enhance fish health and disease resistance has become common practice in aquaculture over the past few decades. This study investigated the effects of hot water extract (HWE)-supplemented feed on serum protein expression in red hybrid tilapia following PAMPs challenge. Fish were fed with a basal or HWE-supplemented diet for 30 days and subsequently injected with lipopolysaccharide (LPS), polyinosinic: polycytidylic acid (poly(I:C)), or PBS (control). Proteomic analysis revealed differential expression of key immune-related proteins like immunoglobulin µ heavy chain and apolipoprotein A-II, which were significantly higher in the HWE-supplemented group upon LPS challenge. On the other hand, C-type lectin domain-containing protein, immunoglobulin µ heavy chain, hemopexin, serotransferrin, α2-HS-glycoprotein-like, inter-alpha-trypsin inhibitor heavy chain H3-like, L-rhamnose-binding lectin, and vitellogenin were lower in the HWE-supplemented diet group as compared to the basal diet group post-poly(I:C) challenge, while plasma protease C1 inhibitor and apolipoprotein Eb increased. Functional classifications analysis revealed that most of the DEPs found were involved in the cellular process, biological regulation, metabolic process, and response to stimulus. LC-MS identified bioactive metabolites in HWE, which may contribute to its immunomodulatory effects. These findings suggest that HWE supplementation modulates immune protein expression and enhances disease resilience in tilapia, offering a sustainable strategy for improving aquaculture productivity.

Molecular Fish Species Identiﬁcation of Commercial Traditional Seafood Products in the Malaysian Market

Fatin Qurratu’aini Zain — 2025-09-30

Seafood is a vital source of protein, multivitamins, and essential fatty acids, particularly omega-3. However, seafood fraud is becoming a growing threat to food safety in Malaysia. This study reports on the molecular identification of fish species in various seafood products collected from different regions in Malaysia. DNA barcoding employing a ~500 bp fragment of the mitochondrial cytochrome c oxidase I (COI) gene was used to identify 46 samples. Successfully amplified and identified samples accounted for 48% of the analysed fish products. Sardinella species were most commonly used in traditional Malaysian products, especially in the east coast states. Interestingly, we found regional variations in fish species used for the same traditional products, highlighting the diversity of local preferences and resource availability. DNA barcoding with the COI marker was found to be effective for the rapid identification of fish species in processed foods, which could enhance food safety and consumer confidence. Although no toxic fish substitutions were detected, the results highlight the need for more comprehensive labelling practices in the Malaysian seafood market. This study emphasises the potential of molecular techniques in addressing food fraud concerns and improving seafood traceability.

Development of pH Indicator Film Containing Butterfly Pea Flower (Clitoria ternatea L.) Extract for Monitoring Sardines and Catfish Freshness During Chilled Storage

Mohammad Saiful Anwar Mohd Yunus — 2025-09-30

An application of pH indicator film has been utilised in food packaging to ensure quality and safety, as well as monitor the real-time freshness of perishable products; however, previous works have focused on synthetic dyes. This research aimed to develop and determine the physicochemical characteristics of a pH indicator film from potato starch incorporated with butterfly pea anthocyanin extracts (PS-BPE) at different concentrations of BPE (4%, 8% & 12%), and evaluate its potential for monitoring the freshness of sardines and catfish. The films were characterised by microstructure, thickness, moisture content (MC), water vapour permeability (WVP), and colour responses at different pH values (2 - 12). Then, the PS-BPE films were applied to fish to monitor the freshness at 4°C for 6 days of storage. The colour changes, pH, texture profile analysis (TPA), and Quality Index Method (QIM) were analysed every two days of storage. The increase in BPE concentration in the film results in an increased thickness and a decreased MC, with no significant difference in WVP of the film. The colour of PS-BPE films also became significantly darker and bolder with an increase in concentration, and colour changes from purple to blue-green were observed during storage. TPA show no difference between the two fish samples. However, both fish increased in pH and QI scores over time, indicating a decline in the quality and freshness of the fish as the storage period extended. These results suggest that PS-BPE films have the potential to serve as a freshness indicator for fish samples.

Astaxanthin Nanoemulsion Pre-Supplementation Mitigates Ischaemic Stroke Injury by Enhancing Neuroprotection and Reducing Infarcted Volume Area

Anis Syahirah Mohd Shafie — 2025-09-30

Ischaemic stroke is the third leading neurological disease that causes death around the world. One of the approaches to reduce severity after an ischaemic stroke is by preparing the brain for neuroprotection. Astaxanthin is a natural product produced by microalgae, Haematococcus pluvialis, against a harsh environment for its survival. It is a well-known carotenoid recognized for its anti-inflammatory and anti-apoptotic benefits. The advancements in nanomedicine of drug design and delivery systems suggest that formulating astaxanthin into a nanoemulsion could enhance its ability to penetrate the BBB and provide neuroprotection, including for ischaemic stroke injury. Rats were assigned to six groups: sham control (SCG), stroke (SG), stroke pretreated with palm olein (SOG), stroke pretreated with astaxanthin extract (SXG), stroke pretreated with astaxanthin macroemulsion (SMG), and stroke pretreated with astaxanthin nanoemulsion (SNG). Supplementation was given orally for 7 days before and once 3 hr after permanent middle cerebral artery occlusion (pMCAO). All groups were subjected to pMCAO for stroke induction except SCG, which received sham surgery. After twenty-four hr, the rats were tested for neurological tests and sacrificed for infarct volume and neuronal markers injuries quantification. This study demonstrated that pre-supplementation with astaxanthin nanoemulsion was able to minimize brain injury caused by ischemic stroke, as evidenced by a low modified neurological severity score, reduced percentage of failures between grids in the grid walking test, and increased latency on the rotarod in the rotarod test. Furthermore, pre- and post-supplementation of astaxanthin nanoemulsion demonstrated a reduction in neuronal cell death as shown by TTC staining and a reduction in neuro-ischemic markers, evidenced by a low level of S100β and NSE in the plasma. In summary, our findings suggest that astaxanthin nanoemulsion could be a promising approach in mitigating the effects of ischemic stroke.

Arabidopsis thaliana Stress Associated Protein 2 (AtSAP2) Expression in Response to Salinity and Drought is Regulated by the RAP2.4 Transcription Factor

Iman Afiq Sakri — 2025-09-30

Plants cannot move to shelter themselves and need to adapt to environmental changes through complex regulatory mechanisms. These adaptations commonly involve the role of stress-related transcription factors (TF). This study explores the role of Ethylene Response Factor (ERF) transcription factor binding sites by examining their distribution within the promoter region of the Arabidopsis thaliana Stress Associated Protein 2 (AtSAP2). Previous studies have highlighted the importance of AtSAP2 in conferring tolerance to plants under abiotic stress, and its ortholog in rice (OsSAP4) has been shown to exhibit increased expression due to ERF binding sites. Thus, understanding the connection between specific ERF sites and the regulation of AtSAP2 is crucial. Analysis of the 2 kb region upstream of the AtSAP2 promoter revealed several Transcription Factor Binding Sites (TFBS), including bZIP, C2H2, CPP, ERF, HD-ZIP, G2-like, MYB, and WRKY. Among these, the Ethylene Response Factor (ERF) binding sites, along with C2H2, are the most abundant in the AtSAP2 promoter. Notably, RAP2.4, an ERF, is significantly predicted to be present in the AtSAP2 promoter (p<0.05). To investigate the relationship between AtSAP2 and RAP2.4 regulation, a promoter study was conducted using Arabidopsis. Two different constructs were generated and transformed into Arabidopsis: Col-0 (pAtSAP2::Col-0) and the rap2.4 mutant (pAtSAP2::rap2.4). The rap2.4 mutant was used to determine whether a mutation in RAP2.4, an ERF, affects AtSAP2 expression through the RAP2.4 binding site. A clear difference in GUS staining activity was observed between pAtSAP2::Col-0 and pAtSAP2::rap2.4. The pAtSAP2::Col-0 plants exhibited blue stain, indicating GUS activity, while no GUS activity was detected in pAtSAP2::rap2.4 plants at either the vegetative (leaf) or inflorescence stage. This suggests that the RAP2.4 protein plays a crucial role in regulating AtSAP2 gene expression through its binding site during abiotic stress.

In Vitro Shoot-Tip Propagation of Blue Peacock Fern Selaginella willdenowii P.Beauv. (Selaginellaceae) an Ornamental Fern

Nurul Nadhirah — 2025-09-30

Selaginella willdenowii, an ornamental fern with iridescent bluish green fronds, is found naturally on forest floors. Many Asian countries consumed it as a traditional medicine and food. It is also suitable for decorative plants since it requires low light and moist conditions, making it perfect for ground covers and house plants. There are currently no efficient methods for propagating this species. Therefore, the goal of this research was to establish an effective in vitro culture propagation method for this species. The shoot-tips were sown in half-strength Murashige & Skoog (MS) as a control, and we observed the most effective culture conditions for shoot-tip proliferation and growth on agar-cultured media at varied concentrations of plant growth regulator (PGR). The findings demonstrated that the culture media supplemented with 0.5 mgL ^-1GA₃ has a positive effect on the shoot tips, forming a maximum number, lengths of the shoots, and numbers of rhizomes with significant difference (ANOVA, ά<0.05). By using this method, it will be possible to speed up the process for growing a large number of S. willdenowii, and it may also be used to propagate other related plants.