Current Issue

 

Volume 27; 2019

 

Effects of methanolic plant extracts on cell proliferation and HIF activity under hypoxic condition in vitro
Pheik-Sheen Cheow, Norazizah Shafee, Sien-Yei Liew, Muhajir Hamid
APJMBB 27 (1): 1-9 
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.01
Click here to download [PDF] [Supplementary Data]
Low oxygen tension is termed as hypoxia. Hypoxia will lead to transcription of hypoxia-inducible factor (HIF) andregulation of downstream gene expression. Underexpression or overexpression of HIF was found to be responsible for various diseases. Proper regulation of this transcriptional factor will aid in treatment of the related diseases. Nowadays, many different approaches are used to modulate HIF, including the usage of naturally-derived plant extracts. Plant extracts are widely accepted compared to other treatments as they are less harmful to the patient and are widely available. In this study, the cytotoxicity of eight different plant extracts under two different gaseous conditions, hypoxic and normoxic, were examined. We also examined the HIF activity shown by the cells under treatment of various concentrations of plant extracts. All eight plants were dried, blended, extracted using methanol, and evaporated to form crude plant extracts. MTT assay was performed by treating the cells with different concentrations of plant extracts and cell viability was determined. Meanwhile, HIF activity of the cells was evaluated by using single luciferase reporter assay. Relative cytotoxicity shown by the cells was different for each plant extract under the various concentration. Pereskia bleo, Orthosiphon aristatus, and Clinacanthus nutans showed high cell viability, 80% of cell viability, within the range of concentration tested. In contrast, Gynura procumbens, Hydrocotyle sibthorpiodies, Pereskia grandifolia, Strobilanthes cripus, and Melastoma malabathricum showed low cell viability. Most of the cells showed activation of HIF activity when treated with different concentrations of the plant extracts. When cells were treated with high concentrations of plant extracts, inhibition of HIF activity were seen and was correlated with low cell viability after treatment. The most notable part of the study was that more than 100% HIF activation was observed for Clinacanthus nutans. However, the cell viability remained high. This might indicate that Clinacanthus nutans is a promising candidate to activate HIF at a transcriptional level with minimal cytotoxicity.
The effect of Malaysian stingless bee, Trigona spp. honey in promoting proliferation of the undifferentiated stem cell
Mohd Amin Marwan Mohamad, Muhammad Alif Mazlan, Muhammad Ibrahim, Afzan Mat Yusof, Shamsul Azlin Ahmad Shamsuddin, Nik Fakhuruddin Nik Hassan, Hussin Muhammad, Muhammad Lokman Md. Isa
APJMBB 27 (1): 10-19 
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.02
Click here to download [PDF]

Stem cells provide various potential applications in regenerative medicine through its ability of self-renewal and differentiation. Among the various stem cells, dental pulp stem cells (DPSCs) have shown encouraging results in their ability to regenerate. Honey has been used in traditional culture as a natural medicine in supporting wound healing. Yet, very few studies on honey were conducted for its potential as a proliferative agent for stem cells. The aim of this study is to evaluate the stability of two Trigona spp. honeys (1 and 2) added in culture media and its proliferative effect on DPSCs. Both honeys were diluted with standard culture medium through dilution process to prepare the concentrations of 0.01%, 0.04%, 0.10% and 0.25%. DPSCs were treated with the diluted honeys for 24 hours. The proliferative activity was determined through the images taken using an inverted microscope for every six hours. In addition, the MTT assay was conducted to determine the cell viability of DPSCs when treated with both honey 1 and 2 at various concentrations. The results showed a stable culture media added with honey for three days and a dose-dependent proliferative effect of both Trigona spp. honey samples on DPSCs. Optimum proliferative effects were observed at 24 hours for both Trigona spp. honey 1 and 2 on DPSCs. The optimum concentration of Trigona spp. honey 1 was from 0.04% to 0.10% and Trigona spp. honey 2 was below 0.01%. It is concluded that Trigona spp. honey has a promising proliferative effect on DPSCs. 

Evaluation of acute and sub-acute oral toxicity of the aqueous extract of Aquilaria malaccensis leaves in Sprague Dawley rats
Redzuan Nul Hakim Abdul Razak, Suzanah Abdul Rahman, Asmah Hanim Hamdan, Roszaman Ramli, Muhammad Lokman Md. Isa, Hussin Muhammad, Nik Fakhuruddin Nik Hassan
APJMBB 27 (1): 20-32
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.03
Click here to download [PDF]
Aquilaria malaccensis or commonly known as ‘gaharu’ is a species of Aquilaria genus and belongs to the Thymelaeaceae family. It is widely distributed in Malaysia, Indonesia, and the Borneo Islands. Traditionally, its leaves were used to relieve bruises and studies have shown that they function as an antioxidant, aphrodisiac, and tranquilizer. Despite its proven beneficial medicinal properties, information regarding its toxicity is limited. Therefore, we performed a safety evaluation on the aqueous A. malaccensis leaves extract (AMAE) in Sprague Dawley rats. The assessment of acute toxicity based on the Organization for Economic Cooperation and Development (OECD) Guideline 420 revealed that AMAE did not influence mortality, clinical appearance, body weight gain, or necropsy findings at a dose of 2000 mg/kg body weight. In the sub-acute toxicity, all doses did not significantly modify the body weight and food and water intake. In male rats treated with 2000 mg/kg, there was a significant reduction in the relative weight of liver. Not only that, an increase in alkaline phosphatase and alanine transaminase was also observed in different groups among the female rats. A significant decrease in the creatinine level was also seen among male rats administered with different doses of AMAE. In both sexes, histopathological analysis had shown abnormalities in the liver and kidney of rats treated at the dose of 2000 mg/kg. In conclusion, the 50% lethal dose (LD50) of AMAE was estimated to be greater than 2000 mg/kg. In sub-acute duration, the findings suggested that AMAE administered orally is slightly toxic at higher doses (2000 mg/kg) and could provoke functional and structural changes in the kidney and liver of rats. Thus, the extract should be used with caution.

Impact of CYP3A4 and CYP3A5 single nucleotide polymorphisms on anastrozole-associated adverse events among Malaysian breast cancer patients
Murtala Abubakar, Huay Lin Tan, Venkata Murali Krishna Bhavaraju, Siew Hua Gan
APJMBB 27 (1): 33-42
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.04
Click here to download [PDF]
The catalytic activity of the cytochrome P450A (CYP3A4) enzyme is reportedly affected by the presence of single nucleotide polymorphisms (SNPs), leading to inter-individual variability in drug efficacy and adverse reactions. CYP3A4 polymorphisms can serve as potential biomarkers for predicting the efficacy of many drugs, including those used in breast cancer treatment. This study was conducted on 94 hormone receptor-positive postmenopausal breast cancer patients who received 1 mg of anastrozole per day. Anastrozole-associated adverse events (AAAEs), such as musculoskeletal adverse events (MSAEs), hot flashes, mood disturbance and vaginal dryness/dyspareunia, were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was performed to determine the allelic frequency of CYP3A4*4, CYP3A4*18A, CYP3A4*18B, CYP3A4*22 and CYP3A5*3. The frequencies of CYP3A4*18A T>C (rs28371759), CYP3A4*18B G>A (rs2242480) and CYP3A5*3 were 0.03, 0.48 and 0.64, respectively. However, no CYP3A4*4 A>G (rs55951658) or CYP3A4*22 C>T (rs35599367) alleles were detected. No significant association was observed between the alleles and the development of AAAEs. We have demonstrated for the first time that CYP3A4*18B G>A is highly prevalent among Malaysian breast cancer patients. The clinical relevance of CYP3A4*18B is currently under investigation by our group.
Expression of cytochrome P450 2C9 (CYP2C9) in Escherichia coli and its functional characterization
Boon Hooi Tan, Yan Pan, Uma Devi Palanisamy, Iekhsan Othman, Nafees Ahmed, Beow Chin Yiap, Chin Eng Ong
APJMBB 27 (1): 43-55
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.05
Click here to download [PDF]
This study aimed to express the major human hepatic drug metabolizing cytochrome P450 (CYP), CYP2C9, together with NADPH cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate its catalytic activities. Co-expression of CYP2C9 and OxR was achieved by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates valsartan and tolbutamide. Results from immunoblotting demonstrated the presence of CYP2C9 protein in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to previously published data. Kinetic parameters, Kmand Vmaxvalues determined from the valsartan and tolbutamide hydroxylase assays, were also concordant with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP2C9 suitable for drug metabolism and interaction studies.
Acute gamma irradiated Stevia rebaudiana Bertoni enhanced particular types of steviol glycosides
Miao Si Chiew, Kok Song Lai, Sobri Hussein, Janna Ong Abdullah
APJMBB 27 (1): 56-65
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.06
Click here to download [PDF]
Stevia rebaudiana Bertoni from Asteraceae family is commercially valuable for its steviol glycosides (SGs) contents, which is 300 times sweeter than commercial sugar. The bottleneck in Malaysia is the lack of suitable stevia varieties that are able to thrive well under her climatic conditions and still produce high SGs. Mutation induction including gamma irradiation is effective in generating genetic variations and developing new plant varieties with desired traits.  This study was aimed to determine the effects of acute gamma irradiation on phenotypic changes and enhancement of SGs contents of Stevia rebaudiana Bertoni variety AKH L1 (herein after will be designated as AKH L1). In vitro shoot tip explants of AKH L1 were subjected to a gamma doses regime of 10Gy to 50Gy, following which phenotypic changes of the irradiated explants and subsequent regenerated plantlets were observed. All irradiated explants exhibited different survival rates, with the lowest at 9.33±8.33% when subjected to 50Gy, while all the control (non-irradiated explants) survived. The LD50 was found to be at 23Gy. Subsequent irradiation of 900 shoot tip explants at 23Gy, produced 468 surviving shoot tips, which were all capable to develop and successfully sub-cultured until the fourth generation, M4. These M4 in vitro mutant plantlets exhibited significant increase in the numbers of leaf (16.07±5.19) and average leaf size (1.12±0.26cm x 0.54±0.15cm). HPLC analysis performed in parallel further revealed the mutant plants contained higher concentrations of stevioside (387.04ppm), rebaudioside A (670.18ppm) and rebaudioside D (106.26ppm) compared to the non-irradiated plantlets, which exhibited 96.87, 194.42 and 28.25ppm, respectively.
Effect of liquid medium on shoots amplification, in vitro flowering and ex vitro rooting of Oldenlandia umbellata L. - a dye yielding medicinal herb
Revathi Jayabal, Manokari Mani, Latha Rasangam, Priyadharshini Selvam, Mahipal Singh Shekhawat
APJMBB 27 (1): 66-74
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.07
Click here to download [PDF]
Oldenlandia umbellata L. gains importance due to its medicinal properties and the presence of anthraquinones based natural dyes in the roots. Present study describes the effect of Murashige and Skoog’s (MS) liquid medium (full strength) on in vitroregeneration, flower bud induction and ex vitrorooting in O. umbellata.  Shoot segments with 2-3 nodes (each node with 2 axillary buds) served as explants for establishment of cultures. The liquid medium augmented with 2.0 mg L-1 6-benzylaminopurine (BAP) with additives (50 mg L-1 of ascorbic acid and 25 mg L-1 each of arginine, adenine sulphate and citric acid) was effective for shoot bud induction (6.4±0.19 shoots per explant within 2-3 weeks). The shoots were further multiplied (89.3±1.07 shoots, 2-3 weeks) when the shoot clusters obtained from the culture initiation directly transferred to the full-strength MS liquid medium incorporated with 1.0 mg L-1 BAP and 0.5 mg L-1 indole-3 acetic acid (IAA) with additives. Flower buds were induced (12.0±0.15 buds per shoot) when the shoots were cultured on 1.0 mg L-1 BAP and kinetin (Kin, 6-furfurylaminopurine) and 0.5 mg L-1 of IAA at 45 µmol m−2s−1 SFPD (Spectral Flux Photon Density) light intensity for 14/10h (light/dark) photoperiod. The adventitious roots were induced on 1/4 strength MS medium supplemented with 1.5 mg L-1 indole-3 butyric acid (IBA). Ex vitro rooting was achieved (16.0±0.53 roots per shoot) by pulse treatment of the shoots with 300 mg L-1 IBA for 2 min. The in vitroproduced plantlets were acclimatized in the greenhouse and finally translocated to thein vivo conditions with 93% success rate. This is the foremost (use of liquid MS medium) and cost-effective method for large scale multiplication of O. umbellata.
Antifungal activities against oil palm pathogen Ganoderma boninense from seaweed sources
Syamimi Diyana Abdul Aziz, Nur Fazirah Jafarah, Suriana Sabri, Mohd Aswad Abdul Wahab, Zetty Norhana Balia Yusof
APJMBB 27 (1): 75-83
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.08
Click here to download [PDF]

Basal stem rot (BSR) disease is the most devastating disease in oil palm which is caused by a fungal pathogen, Ganoderma boninense. However, to date, there is no reliable control for this disease. This study investigated the antifungal potential of seaweed extracts against G. boninense and screening of the compounds possessing this antifungal activity. Four seaweed species namely cfSargassum oligocystumCaulerpa racemosaCaulerpa racemosa var. lamouroxii and cfHalimeda macrophysa were collected from Teluk Kemang, Port Dickson, Malaysia and their antifungal potential against G. boninense were evaluated. Two solvents with different polarities were used for crude extraction namely methanol and chloroform. Antifungal assay using crude methanolic and chloroform extracts from these seaweed species were carried out at various concentrations using the poisoned food technique. Caulerpa racemosa var. lamouroxii chloroform extract showed strong antifungal activity against G. boninense with 27.44% inhibition of the fungus followed by C. racemosa methanolic extract with 26.92% inhibition of the fungus at the lowest extract concentration of 0.25 mg/mL. The extracts were subjected to Gas Chromatography-Mass Spectrometry analysis and the dominant bioactive compounds detected in both extracts were phytol and l-(+)-ascorbic acid 2,6-dihexadecanoate which were also found in plant extracts showing antimicrobial activities in previous studies. The findings suggested that local Malaysian seaweed species have high potential as a source of antifungal compounds which could be useful specifically for the application in the oil palm industry.

Inoculation of fowlpox viruses coexpressing avian influenza H5 and chicken IL-15 cytokine gene stimulates diverse host immune responses
Abdul Razak Mariatulqabtiah, Nadzreeq Nor Majid, Efstathios S. Giotis, Abdul Rahman Omar, Michael A. Skinner
APJMBB 27 (1): 84-94
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.09
Click here to download [PDF]
Fowlpox virus (FWPV) has been used as a recombinant vaccine vector to express antigens from several important avian pathogens. Attempts have been made to improve vaccine strains induced-host immune responses by coexpressing cytokines. This study describes the construction of recombinant FWPV (rFWPV) strain FP9 and immunological responses in specific-pathogen-free (SPF) chickens, co-expressing avian influenza virus (AIV) H5 of A/Chicken/Malaysia/5858/2004, and chicken IL-15 cytokine genes. Expression of H5 (50 kD) was confirmed by western blotting. Anti-H5 antibodies, which were measured by the haemagglutinin inhibition test, were at the highest levels at Week 3 post-inoculation in both rFWPV/H5- and rFWPV/H5/IL-15-vaccinated chickens, but decreased to undetectable levels from Week 5 onwards. CD3+/CD4+ or CD3+/CD8+T cell populations, assessed using flow cytometry, were significantly increased in both WT FP9- and rFWPV/H5-vaccinated chickens and were also higher than in rFWPV/H5/IL-15- vaccinated chickens, at Week 2. Gene expression analysis using real time quantitative polymerase chain reaction (qPCR) demonstrated upregulation of IL-15 expression in all vaccinated groups with rFWPV/H5/IL-15 having the highest fold change, at day 2 (117±51.53). Despite showing upregulation, fold change values of the IL-18 expression were below 1.00 for all vaccinated groups at day 2, 4 and 6. This study shows successful construction of rFWPV/H5 co-expressing IL-15, with modified immunogenicity upon inoculation into SPF chickens.
Characterization of cis-elements in hormonal stress-responsive genes in Oryza sativa
Abbas Saidi, Zohreh Hajibarat
APJMBB 27 (1): 95-102
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.1.10
Click here to download [PDF]
Phytohormones play a key role in plant growth and development. The process of plant’s perception and response to abiotic and biotic stresses is controlled mainly by the phytohormones which act as an endogenous messenger in the regulation of the plant’s status. They can be activated by different signaling pathways in response to environmental stresses. Plants respond to environmental stress through interaction of transcription factors with a handful of cis-regulatory elements (CREs). Some examples of cis elements include abscisic acid-responsive element (ABRE), G-box (CACGTG) element, and W-box. In order to investigate the effects of different hormonal stresses which have a key role in response to biotic and abiotic stresses in rice, microarray data was used. Of the available data, 931 genes revealed significant differences in response to different hormonal stresses such as auxin, cytokinin, abcisic acid, ethylene, salicylic acid, and jasmonic acid. The present results showed that 388 genes were up-regulated, and 543 genes were down-regulated. Most of the genes were up-regulated in response to Indole-3-acetic acid (IAA) hormone. Genes Ontology analysis revealed that they respond to various hormones involved in auxin- responsive genes, auxin-activated signalling pathway and cellular responses to environmental stimuli.G-box had the highest number of cis elements involved in hormonal stress and was regulated by auxin signalling and various stresses. Dehydrin was the only gene up-regulated in response to the six hormones. This gene can be activated in response to abiotic and biotic stresses.As such, dehydrin gene can be used in crop breeding programs to increase tolerance to different environmental stresses in various plant species.
Improvement of microbiological quality, antioxidant content and shelf life of jujube (Ziziphus mauritiana cv. BAU Kul) fruit by gamma irradiation
Farzana Mridha, Roksana Huque, Mst. Afifa Khatun, Mahfuza Islam, Arzina Hossain, Afzal Hossain, Md. Shahinur Kabir
APJMBB 27 (2): 1-9
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.01
Click here to download [PDF]
Postharvest loss of inherently perishable fruits is a matter of serious concern for the farmers and traders. Reduction of postharvest loss is one of the key components for ensuring food security. A study was carried out to reduce the postharvest loss of BAU Kul, an improved variety of jujube fruit (Ziziphus mauritiana), by using gamma irradiation. Different doses of gamma irradiation (0.5, 1.0 and 1.5 kGy) was applied to the jujube fruit samples and the microbiological quality, antioxidant content and shelf life of those fruits were evaluated. Gamma irradiation initially caused significant reduction of the total heterotrophic bacteria, coliform as well as yeast and mold counts. However, the counts increased in both irradiated and non-irradiated fruit samples with the passage of storage period but the increment was significantly less in the 1.5 kGy irradiated samples. Irradiation played active role in the enhancement of total phenolics and flavonoids contents. The concentration of these antioxidants remained higher in irradiated samples in comparison to non-irradiated control samples throughout the storage period. However, the ascorbic acid content decreased gradually with the increase of radiation dose and storage period. The overall acceptability of the fruit samples was determined by the taste-taking panelist. The irradiated (1.0 and 1.5 kGy) fruits were acceptable up to 8 days whereas control and 0.5 kGy irradiated fruits lost their acceptability during storage. The study revealed that 1.5 kGy irradiation can improve microbiological quality and extend the shelf life of jujube fruits (cv. BAU Kul) without significant loss of overall antioxidant content and sensory attributes.
A rhizosphere isolate from Oryza sativa, Enterobacter cloacae VITTPN2, as a potential plant growth promoting rhizobacteria; an in vitro study
Thahiya Naushad, Neethu Kamarudheen, Poorna Chandrika Gopal, Kokati Venkara Bhaskara Rao 
APJMBB 27 (2): 10-17
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.02
Click here to download [PDF]
The increasing need for Plant Growth Promoting Rhizobacteria (PGPR) for biofertilizer development is warranted owing to the environmental hazards caused by chemical fertilizers. Our investigation was to isolate, screen and characterize PGPR from rhizospheric soil with potential PGPR properties. Oryza sativa and Saccharum officinarum rhizosphere were collected from the agricultural research station, Virinjipuram, Vellore (12.9202°N, 79.1333°E), Tamil Nadu, India for PGPR isolation. Eleven distinct isolates of bacteria were grown on Jensen’s (seven) and Pikovskaya’s media (four). Among these, four isolates (TPN1 to TPN4) showed phosphate solubilisation activity. And one isolate TPN2 particularly showed both nitrogen fixation and phosphate solubilization with other PGPR properties. Furthermore, the isolate TPN2 demonstrated promising results in Indole 3-Acetic Acid production (99.29±0.945µg ml-1). Since the isolate TPN2 displayed all PGPR characteristics under study, it was selected for pot culture studies. The seeds treated with TPN2 revealed an increase of 63.6% in shoot length and 14.63% in root length of the okra plant. There was a 74.6% increase in shoot length and a 16% increase in the root length of the tomato plant. Additionally, there was extensive development of lateral roots in okra plant. Henceforth TPN2 was identified as Enterobacter cloacae VITTPN2 (ku951582). This report produced remarkable results which promise the bacterial strain Enterobacter cloacae strain VITTPN2 can be further studied as a prospective biofertilizer.
In vitro antimicrobial assessment on lactic acid bacteria isolated from common freshwater fishes
Wai-Wei Chong, Crystale Siew-Ying Lim, Kok-Song Lai, Jiun-Yan Loh
APJMBB 27 (2): 18-25
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.03
Click here to download [PDF]
Probiotic is well-known as an effective agent to control and manage diseases in aquaculture. Unlike antibiotics and chemotherapeutic agents, probiotic does not trigger the emergence of antibiotic-/chemo-resistant bacteria. This study was aimed to isolate, identify and evaluate lactic acid bacteria from intestines of three common food fish, i.e. tilapia (Oreochromis niloticus), catfish (Clarias gariepinus) and rohu (Labeo rohita). Thirty-four lactic acid isolates were isolated and screened for inhibitory effect against fish pathogens e.g. Escherichia coli, Klebisella pneumoniae, Pseudomonas aeruginosa and Salmonella enterica. Positive antagonists were subsequently tested in haemolytic, salt tolerance and bacteriocin-like inhibitory substances (BLIS) assays. Our results showed only three isolates displayed positive inhibitory effect against all four pathogens. These three isolates were classified as γ-haemolytic bacteria. Our results revealed that bacterial isolates (T2.1.2 - Pediococcus acidilactici and T2.2.2 - Lactobacillus fermentum) isolated from O. niloticus (tilapia) showed a better adaptation in the range of 0-20 ppt; while, the bacteria isolated from L. rohita (R1.1.1 - P. acidilactici) could survive up to 35 ppt. These isolates were then identified based on 16S rRNA gene sequences. BLIS data revealed that both P. acidilactici and L. fermentum isolated from O. niloticus and L. rohita could suppress the growth of pathogens with cell density as low as 10cfu/ml. Our study shows that P. acidilactici and L. fermentum have the potential to be further explored as biocontrol/probiotic agents in aquaculture.
Extraction and preliminary study of antibacterial compounds of three species of Aspergillus genus
Amina Bramki, Meriem Frahtia, Atef Jaouani, Laid Dahimat, Noreddine Kacem Chaouche
APJMBB 27 (2): 26-34
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.04
Click here to download [PDF]
In the interest of discovering  new antibiotic molecules, the antibacterial activity of three fungal strains namely: Aspergillus quadrilineatus, Aspergillus niveus, and Aspergillus wentii isolated from particular ecosystems was sought against six bacterial strains including three with Gram-positive staining (Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis) and three with Gram-negative staining (Escherichia coli, Pseudomonas aeruginosa, Klebsiella  pneumoniae). The results of the agar cylinder technique highlighted that the three fungal strains showed a considerable antibacterial activity. In order to optimize the extraction conditions of the bioactive molecules, five solvents in different polarities were tested, of which chloroform turned out to be the best one. After the selection of this solvent, four culture media of different compositions were used in order to determine the most adequate medium for the production of antibacterial substances. The results revealed that Czapek-dox medium supplemented with yeast extract turned out to be the most favorable one for the production of bioactive molecules from both strains: A. quadrilineatus and A. niveus, while the most suitable medium for the A. wentii strain was Sabouraud. In addition, a study of the antibacterial effect of organic extracts by the Biolog micro-culture system was performed using a range of concentrations. The obtained results revealed that the extracts of the three fungal strains presented a remarkable activity with different concentrations and this, against all the tested bacterial strains. It was recorded only for the three used fungal species, the antibacterial activity was studied for the first time by the Biolog system. 
Characterization of phenol-degrading fungi isolated from industrial waste water in Malaysia
Nadila Hanafee, Nor 'Azzah Mohd Salleh, Siti Aqlima Ahmad, Wan Zuhainis Saad, Mohd Termizi Yusof
APJMBB 27 (2): 35-43
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.05
Click here to download [PDF]
Microorganisms have the ability to degrade phenol. However, in Malaysia, there are lack of study on indigenous microorganisms (fungi) that have the ability to degrade phenol. A total of 141 phenol-degrading fungi isolates were isolated from soil and water samples collected from various industrial areas located in Malaysia. The fungi isolate N12 P6C3 was chosen based on its high efficiency in degrading phenol. The fungi isolate N12 P6C3 isolated from a heavy metal factory, Dungun, Terengganu was able to degrade 700 mg/L of phenol within 6 days and the mycelium growth had increased to 0.25 g. The phylogenetic tree based on the ITS sequence analysis confirmed that the fungal identity was closely related to Penicillium janthinellum strain ATCC 4845. The optimum conditions of this fungus to degrade phenol was attained at temperature of 35°C, ammonium sulphate at 3 g/L, 0.05 g/L of sodium chloride, and pH 6. The ability of P. janthinellum strain N12 P6C3 in the degradation of phenol may provide additional knowledge on locally isolated phenol-degrading fungi which could contribute towards phenol waste management in Malaysia.
Molecular cloning and characterization of NAC genes from four foxtail millet genotypes
Sintho Wahyuning Ardie, Nurul Khumaida, Tetsuo Takano, Nike Karjunita, Muhammad Habib Widyawan
APJMBB 27 (2): 44-49
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.06
Click here to download [PDF]
Transcription factor gene family of NAC (NAM, ATAF, CUC) is tightly involved in plant development and in the response to stresses. In this study, we reported the isolation and the characterization of NAC gene homolog from four foxtail millet genotypes. Band with approximately 1300 bp size was successfully amplified from the genomic DNA of four foxtail millet genotypes (ICERI-4, ICERI-5, ICERI-6 and ICERI-10) using gene specific primer. The fragment was designated as SiNAC065 after showing high similarity with NACgene homologs in the GenBank. Sequence analysis results showed that the SiNAC065 genes isolated from the four genotypes were 1265 bp in length with one intron and two exons. The two exons encode 325 amino acids with the conserved domain located between amino acid 19-325. The SiNAC065 protein identified in this study have 8 conserved motives in the conserved region which categorized them as SNAC (stress responsive NACs) orthologs that are involved in the abiotic stress responses. Different features of SiNAC065 isolated from the tolerant- and the sensitive-genotypes should provide information of the gene’s role in salinity tolerance mechanism of foxtail millet.
Factors contributing to the enhanced production of protease and lipase in Bacillus pumilus SG2 mutant
R. Sangeetha, A. Geetha, I. Arul Pandi
APJMBB 27 (2): 50-55
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.07
Click here to download [PDF]
A random mutagenesis on a wild strain Bacillus pumilus SG2 using physical and chemical agents was performed previously and a mutant Bacillus pumilus SGMM8 which produced comparatively higher levels of protease and lipase was isolated. This study was attempted to analyse the causes underlying the enhanced enzyme production by the mutant. Substrate uptake, repression/de-repression studies and determination of Km and Vmax were done to understand if de-repression and enhanced affinity to substrate are the plausible reasons for enhanced enzyme activity. It was observed that the protease production was not repressed at high glucose concentrations in the mutant. The Km and Vmax of SG2 lipase were 2.265 mg/ml and 158 U/ml respectively and those of SGMM8 lipase were 1.619 mg/ml and 159 U/ml respectively and thus enhanced affinity may be the underlying cause for enhanced lipase activity. The genome sequence of the mutant enzymes were analysed and transversion mutations were identified in the coding regions. The mutations in the signal peptide of the protease and near the active site of the lipase may have caused increased enzyme production/activity.
Identification of polyketide synthase gene clusters in a phage P1-derived artificial chromosome library of a Philippine strain of Streptomyces sp. PCS3-D2
Aileen Bayot Custodio, Edwin Plata Alcantara 
APJMBB 27 (2): 56-63
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.08
Click here to download [PDF]
A phage P1-derived artificial chromosome (PAC) library was constructed from genomic DNA of Streptomyces sp. PCS3-D2. Polymerase chain reaction (PCR) screening of the PAC library revealed two clones, PAC16D and P222O, which were positively identified to harbor polyketide synthase (PKS) Type I and PKS Type III gene clusters, respectively. Restriction enzyme digestion showed that PAC16D and PAC222O contained a 130 kb and a 140 kb insert, respectively. Results of sequencing and bioinformatics analyses revealed that PAC16D comprised of a full-length PKS type I bafilomycin gene cluster while PAC222O harbored truncated siderophore and putative gene clusters as well as a complete PKS III biosynthetic gene cluster. The PKS III gene cluster had three genes similar to alkyl resorcinol biosynthetic genes, however majority of the novel gene cluster had little similarity to known PKS Type III gene clusters. The successful cloning and identification of these gene clusters from Streptomyces sp. PCS3-D2 serve as the jump off point to further genetic manipulation in order to produce the insecticidal natural product in a heterologous host. 
Biosafety of RNA silencing and genome editing technologies in crop plants: Australian and Malaysian research perspectives
Jennifer Ann Harikrishna, Rofina Yasmin Othman, Muhammad Shakirin Mispan, Sadia Iqbal, Yong Han, Michael G. K. Jones
APJMBB 27 (2): 64-69
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.09
Click here to download [PDF]
Research in agricultural biotechnology can produce novel solutions to address the ever growing demand for food, feed, renewable materials and renewable energy using increasingly limited resources. Yet research is expensive with long timelines before implementation can disseminate the benefits to society, so there is a need to maximise co-operation and communication between scientists, stakeholders and their governments, to optimise research, its development and the implementation of research outcomes, into mainstream applications. Recognising the impacts of regulations on biosafety, biosecurity and intellectual property policy on strategies for research, senior and early career researchers from two research intensive universities in Malaysia and Australia, held a workshop to identify and to deliberate over two key areas of technology that offer much promise for agriculture, namely RNA silencing and genome editing. A major focus of the workshop was the regulation of new breeding technologies, and how the regulations need to take into account these new technologies. Themes discussed were the need for harmonisation of international legal frameworks and careful use of terminology, standards and guidelines; and the need for good communication and consensus within and between groups of stakeholders and law-makers. This mini-review highlights the deliberations and recommendations from the workshop.
Genetic identification of Javan hawk eagle (Nisaetus bartelsi) from Indonesia using mitochondrial ROI gene
Suhadi, Dwi Listyorini, Riri Wiyanti Retnaningtyas, Fima Rizki Eka Putri, Dina Ayu Valentiningrum
APJMBB 27 (2): 70-77
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.10
Click here to download [PDF]
Nisaetus bartelsi is a native hawk from Java which its genetic information is not commonly understood yet. As a part of the conservation effort to maintain genetic diversity of this endangered species, this research aimed to obtain COI gene sequences from five individuals of N. bartelsi to confirm its position in the phylogenetic tree. DNA isolation from 5 N. bartelsi blood sample was performed and its COI gene sequence was amplified, sequenced, and used to reconstruct phylogenetic tree using MEGA6 with several other members of NisaetusAquila, and Saggitaridae family. Furthermore, the intraspecific distance between 5 N. bartelsi samples and interspecific distance with other species were calculated using MEGA6. The result suggested that all five individuals belonged to the species Javan hawk-eagle (N. bartelsi) and were closely related to the Blyth’s hawk-eagle (Nisaetus alboniger). The DNA barcoding of the Javan hawk-eagle conducted in this study is a stepping stone to conservation efforts for the Javan hawk-eagle.
Investigating anti-neuroinflammatory mechanism of orientin in lipopolysaccharide-induced BV2 microglia cells
Pei Hong Gan, Erna Laere, Anna Pick Kiong Ling, Kenny Gah Leong Voon, Rhun Yian Koh, Ying Pei Wong
APJMBB 27 (2): 78-92
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.11
Click here to download [PDF]
Chronic neuroinflammation in central nervous system (CNS) can lead to neurodegenerative diseases (ND). This was due to the over-activated microglia, which releases excessive pro-inflammatory mediators. The molecular mechanisms of orientin as anti-neuroinflammatory are yet to be fully elucidated. In order to investigate the effect of orientin on LPS-stimulated BV2 microglial cells, the cells were pre-treated with orientin at maximum non-toxic dose (MNTD) (15 µM) or half MNTD (½ MNTD) (7.5 µM) for 3 hours, followed by incubation with 0.1 µg/mL of LPS for 24 hours. The LPS-stimulated cells were then subjected to three series of studies, including the determination of ROS level using 2’,7’-dichlorofluorescindiacetate (DCFH-DA) methods and the determination of mRNA of nuclear factor (NF)-кB, Signal transducer and activator of transcription 1 (STAT1), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) via real-time PCR (qPCR). The findings from this study demonstrated the probable mechanism of orientin in treating neuroinflammation via the downregulation of ROS level, STAT1, NF-кB, iNOS and COX-2 whilst upregulating HO-1. Validation of molecular mechanism of orientin suggested that it could be a potential therapeutic agent in treating ND.
Improved extracellular excretion of β-cyclodextrin glycosyltransferase from Escherichia coli by glycine supplementation without apparent cell lysis
Nik Ida Mardiana Nik-Pa, Suraini Abd-Aziz, Mohamad Faizal Ibrahim, Noorjahan Banu Mohamed Alitheen, Norhayati Ramli
APJMBB 27 (2): 93-102
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.12
Click here to download [PDF]
The use of an effective inducer feeding strategy without causing cell lysis presents significant advantage to enhance the secretion of an enzyme to the culture medium of Escherichia coli. The cgt gene encoding β-cyclodextrin glycosyltransferase (β-CGTase) was cloned into pQE30xa as an N-terminal His-tagged protein and transformed into E. coli. The induction strategy was applied towards enhancing the extracellular secretion of the recombinant β-CGTase by increasing permeability of the outer membrane of E. coli. The supplementation of 1.2 mM glycine following 2 h of fermentation at 37°C enhanced the activity of β-CGTase to 38.295 U/mL, which was approximately 1.3-fold higher than the control (without induction). Further flow cytometry analysis was adopted as a rapid and highly reproducible approach to determine the effect of glycine supplementation on the viability of E. coli cells. The supplementation of glycine did not contribute to apparent cell lysis, with no adverse effects on cell viability, hence indicating the effectiveness of glycine in enhancing the extracellular secretion of β-CGTase. The recombinant β-CGTase was then purified through a combination of diafiltration and Ni-NTA affinity chromatography with 18.4-fold increase in purity. An effective glycine feeding strategy could enhance the extracellular secretion of β-CGTase without adverse effects on cell viability. This strategy could be applied potentially to enhance the secretion of a recombinant protein to the culture medium from E. coli cells without having cell lysis. 
Phylogenetic patterns in the tribe Acacieae (Caesalpinioideae Fabaceae) based on rbcL, matK, trnL-F and ITS sequence data
Aramide Dolapo Igbari, Oluwatoyin Temitayo Ogundipe
APJMBB 27 (2): 103-115
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.2.13
Click here to download [PDF] Supplementary Data [1] [2]
The tribe Acacieae is one of the three tribes of the distinct mimosoid clade nested within the re-circumscribed sub-family Caesalpinioideae. Manyuncertainties exist with the taxonomic status of tribe Acacieae in relation to tribe Ingeae andgenus
Acacia. To unravel the phylogenetic patterns within Acacieae, nine members of the tribe were phylogenetically analysed employing both parsimony and Bayesian methods. Six data matrices (ITS, rbcLmatKtrnL-FrbcL+matK+trnL-F and ITS+rbcL+matK+trnL-F) representing 46 sequences, and 2 outgroup taxa were used for the analysis. Our results are in support to some previous studies on the phylogeny of the Acacieae. It supports the polyphyly of tribe Acacieae. The monophyly of VachelliaSenegalia and Faidherbia taxa were strongly supported at >70% bootstrap support values and >0.90 bayesian inference. An unresolved basal paraphyletic clade of Acacia auriculiformis with the outgroup taxa was shown in all the datasets, at mostly low support values. Faidherbia albida was nested within the Senegalia grade while A. auriculiformis (Acacia s.s.) was the closest taxon to the outgroup taxa. A key finding of this study is the polyphyly of Albizia and its close association with A. auriculiformis. 
The identification of SNPs in THCA synthase gene from Pakistani Cannabis
Saadat Ali, Mehreen Mufti, Mehwish Khan, Ishrat Aziz
APJMBB 27 (3): 1-9
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.01
Click here to download [PDF]
Five different isolates of Pakistani cannabis belonging to varying locations were analyzed for the presence of a reported tetrahydrocannabinolic acid (THCA) synthase marker or gene. The amplification of the marker (12 kb) from the five isolates confirmed them to be drug-type since the association of the marker with drug-type cannabis plants had already been established in other reports. The sequence analysis of the THCA synthase marker revealed two single nucleotide polymorphisms (SNPs) (i.e. A851→T851 and A883→C883) specific to Pakistani drug-type cannabis. Furthermore, the predicted protein sequence of the isolated sequences also showed two amino acid substitutions (D284→V284 and T295→P295) corresponding to the identified SNPs. However, the homology based three dimensional models of the inferred proteins generated via Swiss-Model-an automated online server did not project any changes at the active sites of the enzyme (THCA) due to D→V and T→P substitutions. The two missense mutations uncovered as a result of this study may assist in distinguishing the products of Pakistani cannabis among the smuggled materials.
Two-dimensional electrophoresis protein profiles of HL-60 and CCRF-CEM cell lines treated with epigenetic modification drugs
Aziee Sudin, Haiyuni Mohd Yassim, Shafini Mohamed Yusoff, Shaharum Shamsuddin, Ridhwan Abdul Wahab, Muhammad Farid Johan
APJMBB 27 (3): 10-23
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.02
Click here to download [PDF]
Leukemia is classified as a malignant disease of hematopoietic stem cells (HSCs) that failsin cell differentiation but preserve their self-renewal. It is caused by genetic alterations and epigenetic modifications resulting in the activation or inactivation of particular genes for transcription. Epigenetic causes changes in gene expression without any alteration in the DNA sequence. The most common epigenetic modifications are DNA methylation and histone acetylation. 5-Azacitidine (5-Aza) is a DNA methytransferase inhibitor (DNMTi) that inhibits DNA methyltransferase enzymes resulting in hypomethylation. Trichostatin A (TSA) is a histone deacetylase inhibitor which inhibits deacetylation of both histone and non-histone proteins resulting in chromatin relaxation. This present study focused on the alteration of proteome profile on 2D gel electrophoresis (2-DE) induced by 5-Aza and TSA in HL-60 and CCRF-CEM cell lines as in vitro model to represent acute promyelocytic leukemia (APL) and T-lymphoblastic leukemia (T-ALL), respectively. Total proteins of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM cell lines were extracted using urea/thiourea buffer and stained with Coomassie Blue. Comparative analysis of untreated and 5-Aza/TSA-treated HL-60 and CCRF-CEM was performed by PDQuest software. Qualitative analysis identified 190-659 protein spots detected in untreated, 5-Aza and TSA-treated HL-60 and CCRF-CEM. Quantitative comparison analysis was analyzed by over 2-fold change in 5-Aza/TSA-treated cells compared to untreated. One and eight upregulated proteins were detected in 5-Aza and TSA-treated HL-60, respectively. While five and one upregulated proteins were detected in 5-Aza and TSA-treated CCRF-CEM, respectively. These preliminary results suggested that 5-Aza and TSA induced proteome profiles alterations due to their inhibition effects in HL-60 and CCRF-CEM cell lines.
Acute toxicity of glyphosate on various life stages of calanoid copepod, Pseudodiaptomus annandalei
Xue-Er Lim, Kok-Song Lai, Hon-Jung Liew, Jiun-Yan Loh
APJMBB 27 (3): 24-31
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.03
Click here to download [PDF]
Copepods are one of the most important primary producers and biodiversity indicators. They are also highly susceptible to various toxicants. In this study, glyphosate (Roundup®), a widely used herbicide was used to investigate the toxicity effect on calanoid copepods, Pseudodiaptomus annandalei, focused on their nauplius, copepodid, and adult stages. Different concentrations of glyphosate (i.e. 0 - as control, 0.05, 0.1, 0.4, 1.6, 6.4 and 25.6 mg/L) were used to elucidate the tolerance level of P. annandalei. The survival rate of copepodwas recorded at the intervals of 24, 48, 72 and 96 h after glyphosate exposure. The analysis was performed using probit test to determine the sub-lethal concentrations. Our results revealed that LC50 of the nauplius stage was recorded as 3.47, 3.02, 1.86 and 1.10 mg/L at 24, 48, 72 and 96 h, respectively. Higher LC50 values were recorded at 4.36 mg/L for 24 h, 3.09 mg/L for 48 h, 2.00 mg/L for 72 h, and 1.12 mg/L for 96 h at the copepodid stage. Generally, adult copepods showed a higher level of tolerance to glyphosate among all stages, whereby at this stage LC50 values were recorded as 11.70 mg/L for 24 h,10.23 mg/L for 48 h, 7.41 mg/L for 72 h, and 3.61 mg/L for 96 h, respectively. Our results indicated that prolong exposure time of glyphosate could increase the susceptibility of P. annandalei to the herbicide. Nauplii are the most sensitive group among all.This study showed that glyphosate could post significant eco-toxicological impact to the non-targeted organism.
Background correction method for DNA microarray image processing
Omar Salem Baans, Asral Bahari Jambek
APJMBB 27 (3): 32-38
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.04
Click here to download [PDF]
Most microarray image scanning approaches provide an estimation of the intensity of the foreground and background for each spot. Background intensity must be corrected in order to remove the effect of non-specific binding or spatial heterogeneity across the array, but when such corrections are applied many problems appear, such as negative intensity for the spot or high variability of low-intensity log ratios. In this paper, many alternative methods for calculating background intensity are discussed and many approaches for background correction are tested and compared. GenePix, ScanAlyze and QuantArry are the strategies that were reviewed for background locations to extract their intensity. Similarly, to GenePix, a new approach for background calculation was proposed and tested. It shows more accurate results and the occurrences of error become lesser.
Application of compost in mixed media improved oil palm nursery's secondary root structure thereby reducing the fertilizer requirement for growth
Siti Suliza Salamat, Mohd Ali Hassan, Yoshihito Shirai, Ahmad Husni Mohd Hanif, Izwanizam Arifin, Mohamad Shahkhirat Norizan
APJMBB 27 (3): 39-49
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.05
Click here to download [PDF]
Although conventional inorganic fertilizers increased plant growth and productivity, their excessive use leads to wastage, run-offs and environmental pollution. In order to promote nutrient recycling and zero emission strategy in the palm oil industry, compost was produced from oil palm empty fruit bunch (EFB) and palm oil mill effluent (POME) anaerobic sludge. The main objective of this study is to determine the effect of compost in the mixed media on the growth and inorganic fertilizer requirement in the oil palm nursery. 100% soil as media with 100% inorganic fertilizer as control was compared with mixed media containing 50% compost in soil with inorganic fertilizer at 25%, 50%, 75% and 100% compositions and tested for plant growth and root structure. The results showed that the treatments with compost addition improved plant growth, compared to the control with 100% inorganic fertilizer which did not contain compost in the mixed medium. The improved plant growth corresponded directly to the enhanced secondary root structure, which probably resulted in more efficient absorption and uptake of nutrients by the plants. Furthermore, the plant growth and the secondary root structure in the mixed media with 50% inorganic fertilizer composition was not significantly different to the media with 75% and 100% inorganic fertilizer. Therefore it is suggested that the application of 50% compost in the mixed media enhanced the secondary root structure, resulting in reduced inorganic fertilizer requirement in the oil palm main nursery, without affecting the plant growth.
Immunoreactivity of estrogen receptor alpha in brain and ovary of the short mackerel Raestrelliger brachysoma (Bleeker, 1851)
Sinlapachai Senarat, Jes Kettratad, Niwat Kangwanrangsan, Wannee Jiraungkoorskul, Francis Gerald Plumley, Masafumi Amano, Akio Shimizu, Piyakorn Boonyoung, Gen Kaneko
APJMBB 27 (3): 50-63
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.06
Click here to download [PDF]
The reproductive physiology concerning the gonadotropin hormone (GTH) and its downstream target estrogen receptor alpha (ERα) is not well understood in the short mackerel Rastrelliger brachysoma, an economically important marine fish in Thailand. In this study, we tested the hypothesis that the expression of  both GTH and ERα in the brain and ovary of R. brachysoma was as reported in other fish species. By applying immunohistochemical techniques, we identified the distribution of ERα-immunoreactive (ir) neurons in the brain and ovary of wild female R. brachysoma during the spawning season along with the distribution of GTHs-ir cells in the ovary. The nucleus lateralis tuberis in the diencephalon had a high number of ERα-ir neurons. In the mesencephalon, dense ERα-ir neuronal fibers were mainly found in the mesencephalic cells, stratum opticum, stratum fibrosum et griseum superficiale and stratum album centrale. Both the valvula and corpus cerebelli in the metencephalon contained ERα-ir neurons in granular and Purkinje cell layers as well as the molecular layer. The ERα-ir neurons were also observed in the medulla oblongata. In the ovary, weak ERα and moderate GTHs immunoreactivities were observed in follicular cells of oocytes in early and late vitellogenic stages. This information provides baseline data required to understand not only the activity of estrogen (E2) on the brain but also the regulatory mechanism of the hypothalamo-pituitary-ovarian axis of R. brachysoma.
Engineering of Bacillus megaterium for improving PHA production from glycerol
Javier Ricardo Gómez, Rodrigo Velasco Bucheli, Carlos del Cerro Sánchez, Isabel de la Mata Riesco, Amanda Lucía Mora Martínez
APJMBB 27 (3): 64-72
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.07
Click here to download [PDF] [Supplementary Data]
There are a few PHA-producer bacteria that can uptake glycerol to produce this biopolymer. Among them, Bacillus megaterium LVN01 has demonstrated to be able to grow up using glycerol as a carbon source. Glycerol dehydrogenase (GD) plays a key role in the synthesis of PHA from glycerol. In this study, the improvement of glycerol uptake by a recombinant strain of B. megaterium carrying pHT01-bmgd was evaluated in order to enhance PHA production. The biomass and PHA production were evaluated and compared to wild-type. It was determined that the PHA produced by both strains was PHB and the highest improvement in PHB yield was 226% at 30 h.
V protein, the virulence factor across the family of Paramyxoviridae: a review
May Ling Tham, Khatijah Yusoff, Sarah Othman, Suet Lin Chia
APJMBB 27 (3): 73-85
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.08
Click here to download [PDF] [Supplementary Data]
Paramyxoviridae is a family of viruses within the order Mononegavirales and comprises 14 genera; Metaavulavirus, Orthoavulavirus, ParaavulavirusSynodonvirus, Ferlavirus, Aquaparamyxovirus, HenipavirusMorbillivirus, Respirovirus, Jeilongvirus, NarmovirusSalemvirus, Pararubulavirus and Orthorubulavirus. The members within this family are negative and single-stranded RNA viruses including human and animal pathogens such as measles virus (MeV), Nipah virus (NiV), mumps virus (MuV), Sendai virus (SeV) and Newcastle disease virus (NDV). The V protein is conserved within the family and plays an essential role in viral pathogenicity. Although V proteins of many paramyxoviruses are interferon-antagonists which counteract with the host’s innate immunity, there are still differences in the mode of action of the V protein between different genera or species within the same genera. The strategies to circumvent the host interferon (IFN) pathway can be divided into three general mechanisms; degradation of signal transducers and activators of transcription (STAT) protein, inhibition of phosphorylation of the transcription factor and, inhibition of translocation of STAT proteins into the nucleus. As a result, inhibition of IFN signalling and production promotes viral replication in the host cells. This review highlights the mechanism of the paramyxoviral V protein in evading the host IFN system.
An update on the detection methods of Parachlamydia acanthamoebae, an atypical agent of pneumonia
Avinash Rames
APJMBB 27 (3): 86-100
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.09
Click here to download [PDF]
Parachlamydia acanthamoebae (P. acanthamoebae) has been recognized as an emerging agent of pneumonia as it has been identified in human samples via culture-based, molecular and serological techniques. Additionally, studies on animal models have shown that it fulfills the third and fourth Koch postulates to be assigned a pathogenic role. Due to the threat posed by it, multiple tools have been employed in the search for P. acanthamoebae. The methods utilized for its detection would be cell culture based approaches which involve both animal and amoebal cell culture and also molecular techniques that encompasses polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and in situ hybridization (ISH). Additionally, immunohistochemistry (IHC) and serology based techniques such as direct and indirect immunofluorescence are also employed with the usage of Western blotting or immunoblotting as confirmatory procedures. This review attempts to describe the variety of techniques that are present in literature for the isolation and identification of P. acanthamoebae. 
Standardized bioassays: An improved method for studying Fusarium oxysporum f. sp. cubense race 4 (FocR4) pathogen stress response in Musa acuminata cv. 'Berangan'
Yusmin Mohd-Yusuf, Norzulaani Khalid, Jameel R. Al-Obaidi, Nadiya Akmal Baharum, Kamilatulhusna Zaidi, Baharuddin Salleh, Nurul Farizah Azuddin, Fashli Aziz Abdul Aziz, Umaiyal Munusamy, Rofina Yasmin Othman
APJMBB 27 (3): 101-112
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.10
Click here to download [PDF]
To date, there is no standardized Fusarium bioassay protocol established owing partly to the wide variety of Fusarium oxysporum f. sp. cubense (Foc) isolates and banana cultivars present. Thus, validation of the infection parameters is deemed essential prior to each bioassay experiment. In the current study, a simple standardized workflow was developed based on available assays for testing Fusarium wilt disease response in Musa acuminata using M. acuminata cv. ‘Berangan’ of tissue-culture origin as a model. The phenotypic assays were able to detect external disease symptoms less than one week post-inoculation, while the molecular approach using RT-qPCR identified differential expression of catalase (CAT), pathogenesis-related 10 (PR10), phenylalanine ammonia-lyase (PAL) and xylanase (XYL) genes as early as day 0. The transcript levels of PR10 and XYL fluctuated over 4 days of Foc Race 4 (FocR4 C1 HIR isolate) infection while the expression of CAT steadily increased over time. In contrast, PAL was highly upregulated at 2 days post-inoculation. These signature changes suggest that all genes tested might be involved in the early defense response of ‘Berangan’ plants against FocR4 infection. ‘Berangan’ cultivar was found to be highly susceptible to Foc Race 4 (C1 HIR isolate) with leaf symptoms index (LSI) and rhizome discoloration index (RDI) scores of 4.257 and 5.971, respectively. The procedure elaborated in this study can be used as a reference Foc bioassay for reproducible and comparable results possibly across cultivars and test isolates due to its simple steps aided by integration of phenotypic and molecular approach
In silico analysis of signal peptides or secretory production of α-amylase in Bacillus subtilis
Marzieh Asadi, Mortaza Taheri-Anganeh, Zeinab Jamali, Seyyed Hossein Khatami, Cambyz Irajie, Amir Savardashtaki, Younes Ghasemi
APJMBB 27 (3): 113-124
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.3.11
Click here to download [PDF]
α-Amylases are important commercial enzymes and have a broad application in industrial processes and medicine. Gram-positive bacteria such as Bacillus subtilis are possible host organisms for α-amylases secretory production. Secretion of α-amylases to the culture medium versus intracellular production has several advantages such as prevention of inclusion bodies accumulation, higher product stability and solubility. Signal peptides are considered as one of the most essential elements for successful secretory synthesis of the recombinant proteins. Therefore, by the selection of an efficient signal peptide, secretion of the recombinant protein can be enhanced. The goal of this investigation was the In silico evaluation of several peptides to find the most suitable leader peptides for secretory production of α-amylase in B. subtilis. In present work, 30 signal peptides were selected, and numerous online servers such as SignalP, ProtParam, SOLpro, PRED-TAT and ProtComp was used for investigation of suitable signal peptides. According to in silicopredictions all other signal peptides connected to α-amylase were stable and soluble except PPBD_BACSU. PPBD_BACSU because of having D-score below cut-off could not be recognized as a suitable signal peptide for α-amylase. Computational analysis identified QOX2_BACSU may direct protein into transmembrane location and was ignored. All 28 remained were predicted as secretory signal peptides which can excrete protein out of the bacteria. The signal peptides recommended by the present study are valuable for rational designing of secretory soluble α-amylase. Although, such information can be useful for future experimental production of these mentioned secretory proteins.
Characterization of thermostable aminoacylase from Geobacillus sp. strain SZN
Suzana Adenan, Chee Fah Wong, Haniza Hanim Mohd Zain, Saripah Salbiah Syed Abdul Azziz, Raja Noor Zaliha Raja Abd. Rahman 
APJMBB 27 (4): 1-9
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.01
Click here to download [PDF] [Supplementary Data]
Aminoacylase (EC 3.5.1.14) hydrolyzes N–acetylated amino acids to produce amino acids. Although thermostable aminoacylase has been commercially produced since 2004, there was a knowledge gap in the field of understanding aminoacylase thermostability from a structural point of view. This study investigated the physical and structural properties of the purified thermostable aminoacylase SZN. The spectropolarimetry data for structural determination has indicated a gradual decrease of α-helix from 36 to 27.6%, followed by tremendous disorientation of the structure at the transition of temperatures from 60 to 70°C (27.6 to 19.5%). In contrast, the percentage of β-sheet has increased steadily over the tested temperatures. The α-helix, where notable metal binding and catalytic residues are located, was totally weakened at temperatures above 70°C, thus resulted in loss of activity. The loss of the α-helical structure could further explain drastic deterioration of activity at temperatures beyond 70°C. The activity of aminoacylase SZN was enhanced by divalent metal ions, such as Mn2+ and Cu2+, and inhibited by detergent Triton-X-100. As a conclusion, the isolated aminoacylase SZN was characterized as a thermostable enzyme based on the α-helical structure integrity and functional stability in high temperatures. This enzyme could be used as an alternative enzyme for bioindustries in view of its activity enhancement in high temperatures and stability in various tested inhibitors.
Effect of fungal filtrates on germination of wheat grains and the biological control of these fungi using black pepper extract
Fuzia Elfituri Muftah Eltariki, Abdulmajeed Bashir Melitan, Seok Mui Wang, Mohammed Abdelfattah Alhoot
APJMBB 27 (4): 10-18
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.02
Click here to download [PDF]
Wheat is one of the world's most abundant and essential food crops. It covers a significant area of the earth's surface, higher than any other plant, and tends to be among the top strategic crops. Wheat contamination with fungi leads to rapid deterioration of quantity and quality of wheat products. Many of those fungi are potential mycotoxins producers. This study aimed to isolate and identify fungi that contaminating the wheat grains from the Misurata Agricultural Research Center area and the South Region of Libya. Fungi contaminating wheat grains were isolated on Potato Dextrose Agar (PDA) and identified by culture characteristics and microscopically. Fungal filtrates of two fungal isolates, Aspergillus niger and Rhizopus sp., were tested for their effects on the germination and seedlings of wheat grains. Furthermore, the effect of acetonic extracts of Black pepper (Piper nigrum) on the growth of the isolated fungi was also investigated. Ten types of fungi belonging to four genera were isolated and identified. The germination rate of wheat grains irrigated with the filtrate of A. niger and Rhizopus sp. was 20% and 80% respectively, compared with 100% of the control grains, which were irrigated with water. The culture filtrates of both A. niger and Rhizopus sp. affect not the only percentage of grains germination but also the morphology of wheat seedlings. It adversely affected the length of the radicles and coleoptiles. The acetone extract of P. nigrum showed inhibitory effect (85.7% ± 3.7 and 44.0% ± 3.1) on the germination of A. niger and Rhizopus sp. respectively. This study concludes that fungal secretions have pathogenic effects on plant growth, which can lead to potential health risks for the human population. Biological control such as Piper nigrum extracts can be an alternative to chemical pesticides for controlling fungal pathogens and their secretions. 
Determination of potential solvents for novel N-substituted 5-(phenylamino)uracil derivatives and evaluation of their cytotoxic effects on Vero 76 cell
Nur Fahitah Abu Hanipah, Noor Farah Omar Ahmad, Minaketan Tripathy, Elena Gureeva, Michail Novikov, Yulia Gushchina, Olga Butranova, Nafeeza Hj Mohd Ismail, Seok Mui Wang, Anna Krasilnikova
APJMBB 27 (4): 19-29
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.03
Click here to download [PDF]
N-substituted 5-(phenylamino)uracil derivatives have recently shown to possess potential antiviral properties. However, the high lipophilicity of these compounds has limited their ability to be dissolved in aqueous media for further in vitro and in vivo studies. This study aimed to determine the potential solvents for novel N-substituted 5-(phenylamino)uracil compounds and to evaluate the cytotoxic effects of these solvents on Vero 76 cells. Eight solvents, namely acetone, methanol, ethanol, dimethyl sulfoxide (DMSO), polyvinylpyrrolidone, nicotinamide, L-arginine, and sodium benzoate, were used to dissolve 1600 µM each of compound Z214 and compound Z276, which were chosen as the representatives of novel N-substituted 5-(phenylamino)uracil derivatives. Only L-arginine (700 mM), sodium benzoate (1500 mM), and DMSO (128 mM) were able to solubilise both compounds. Cytotoxicity assays on Vero 76 cells have shown that the maximum concentrations of L-arginine, sodium benzoate, and DMSO that demonstrated 100% cell viability were 108 mM, 10 mM, and 211 mM respectively. L-arginine at concentrations ranged from 215 mM to 860 mM have shown to significantly increased cell proliferation; while both sodium benzoate and DMSO have significantly reduced cell viability at concentrations ≥ 10 mM and ≥ 211 mM respectively. CC50 values were 23.22 mM and 214.92 mM for sodium benzoate and DMSO respectively. The findings in this study revealed that DMSO at a concentration of 211 mM was found to be the most appropriate solvent to solubilise 1600 µM and below of novel N-Substituted 5-(phenylamino)uracil derivatives.
Analysis of normalization method for DNA microarray data
Omar Salem Baans, Asral Bahari Jambek, Khairul Anuar Mat Said
APJMBB 27 (4): 30-37
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.04
Click here to download [PDF]
Normalization is a process of removing systematic variation that affects measured gene expression levels in the microarray experiment. The purpose is to get more accurate DNA microarray result by deleting the systematic errors that may have occurred during the making of DNA microarray Image. In this paper, five normalization methods of Global, Lowess, House-keeping, Quantile and Print-tip are discussed. The Print Tip normalization was chosen for its high accuracy (32.89 dB and its final MA graph shape was well normalized. Print tip normalization with PSNR value of 33.15dB has been chosen as a new normalization method. The results were validated using four images from the formal database for DNA microarray data. The new proposed method showed more accurate results than the existing methods in term of four parameters: MSE, PSNR, RMSE and MAE.
Plamodial enzymes in metabolic pathways as therapeutic targets and contemporary strategies to discover new antimalarial drugs: a review
Nurhainis Ogu Salim, Noor Azian Md Yusuf, Fazia Adyani Ahmad Fuad
APJMBB 27 (4): 38-53
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.05
Click here to download [PDF]
Malaria continues to pose imminent threat to the world population, as the mortality rate associated with this disease remains high. Current treatment relies on antimalarial drugs such as Artemisinin Combination Therapy (ACT) are still effective throughout the world except in some places, where ACT-resistance has been reported, thus necessitating novel approaches to develop new anti-malarial therapy. In the light of emerging translational research, several plasmodial targets, mostly proteins or enzymes located in the parasite’s unique organelles, have been extensively explored as potential candidates for the development of novel antimalarial drugs. By targeting the metabolic pathways in mitochondrion, apicoplast or cytoplasm of Plasmodium, the possibility to discover new drugs is tremendous, as they have potentials as antimalarial therapeutic targets. This literature review summarizes pertinent information on plasmodial targets, especially enzymes involved in specific metabolic pathways, and the strategies used to discover new antimalarial drugs.
Cytogenotoxic effects of cypermethrin on root growth: Allium sativum as a model system
Temitope Olabisi Onuminya, Tochukwu Emmanuel Eze
APJMBB 27 (4): 54-61
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.06
Click here to download [PDF]
Cypermethrin is a pyrethroid pesticide used on agricultural farms in Nigeria to control pests of fruits and vegetables but this is highly toxic to aquatic organisms. To determine toxicity of chemicals, Allium cepa is commonly used as an established bioassay however the bulb is used whole. Allium sativum in contrast is able to be split into cloves of smaller units hence this research aims to validate the potential of Allium sativum as a model plant for genotoxicity assessment. In this study, root growth inhibition test and chromosome aberration assay were used and the effective concentration (EC50) of cypermethrin was determined from the root growth curve. Furthermore, the mitotic activities of the root meristem were assessed using light microscopy. Treatment of root meristem of A. sativum with various concentrations of cypermethrin (0.25, 0.50, 0.75 and 1.0 mg/ml) revealed a reduction in the root length and EC50 of 0.44 mg/ml. Also morphological changes such root wilting, dark spots, tenderness of the clove bases and discoloration of the roots were observed. Cytological studies showed a reduction in mitotic index with increasing cypermethrin concentration. Chromosomal aberrations ranging from abnormal metaphases: c-metaphases, disturbed spindles and vagrant metaphases; to abnormal anaphases: laggard chromosomes, chromosome breaks and multipolarities were also recorded. These aberrations reduced with increased concentration of the pesticide leading to the production of lesser number of dividing cells. These show that cypermethrin is genotoxic to the root meristem and A. sativum is a suitable model plant for detecting pyrethroid genotoxicity in plant.
Identification of anti-inflammatory compound/compounds in hexane fraction of Jatropha curcas root extract
Ahmad Razi Othman, Intan Safinar Ismail, Norhani Abdullah, Syahida Ahmad
APJMBB 27 (4): 62-68
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.07
Click here to download [PDF]
Jatropha curcas is a medicinal plant with many therapeutic properties such as anti-inflammatory, anti-malaria, anti-cancer and antioxidant. The root extract has been shown to possess high anti-inflammatory activity. Previously, the compounds responsible for this activity have not been fully elucidated. Two fractions (Fraction 1 and Fraction 2) obtained from a preparative HPLC of the root extract showed significant anti-inflammatory and cytotoxic activities in RAW 264.7 murine macrophage cells with Fraction 1 giving higher nitric oxide (NO) inhibition compared to Fraction 2 and L-NAME. Further purification steps involving column chromatography, thin layer chromatography and analytical HPLC of Fraction 1 produced two fractions labeled as Fraction A and Fraction B. Both fractions showed anti-inflammatory activity without cytotoxic activity in RAW 264.7 cells. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis showed that Fraction A contained a group of 18 carbon fatty acid compounds consisting of 2 oxooctadecanoic acids; 15, 16 dihydroxy 9Z, 12Z octadecadienoic acid; octadecadienoic isomer and 15,16 dihydroxy 9Z, 12Z octadecadienoic acid, 15S, 16S. The 18-carbon fatty acid structure was confirmed by nuclear magnetic resonance (NMR) spectral data. The IC50 value of compounds in Fraction A for anti-inflammatory activity in RAW 264.7 cell line was 434.8±0.75 µg/mL. From the analysis, it can be concluded that Fraction A can be classified under 18 carbon long chain fatty acid group based on LC MS/MS and NMR analysis. This active compound shows an inhibition towards NO activity.
Molecular identification and characterization of medically and veterinary important flies of Bangladesh based on mitochondrial COI gene sequences
Faria Farhana Rain, Abdul Jabber Howlader, Abu Faiz Md. Aslam
APJMBB 27 (4): 69-79
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.08
Click here to download [PDF]
Flies are considered serious pests which cause health problems of human and animal, transmitting many pathogenic microbes. Pest management programs depend on proper identification of pests. The present research work is an initiative to identify the medically and veterinary important flies based on mitochondrial COI gene sequences. Eleven species of the fly pests were identified. Among them, four fly species were the first record from Bangladesh. The phylogenetic analysis of retrieved sequences confirmed that the evolution of these species occurred from a common ancestor. Highest AT percentage (69.9%) was found in Haematobia irritans exigua and lowest GC percentage (30.4%) was found in Haematobia irritans exigua. The substitution rate of codon was found 1.88 in 1st position, 0.73 in 2nd position and 1.22 in 3rd position, respectively. Interspecific genetic divergence range of flies sequences was 5-20%. Haplotype network showed that Atylotus agrestis was mostly diverged from its common ancestors by 37 mutational steps. This research is the first molecular approach to identify the medically and veterinary important flies based on MT-COI gene sequences along with the establishment of first DNA barcode dataset for accurate identification in Bangladesh. 
Synthesis and characterisation of chitosan nanoparticle as a potential delivery carrier
Noor Hidayah Ibrahim, Mas Jaffri Masarudin, Abdul Rahman Omar, Mohd Hair Bejo, Raha Abdul Rahim, Nurulfiza Mat Isa
APJMBB 27 (4): 80-84
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.09
Click here to download [PDF]
Chitosan is a biodegradable, non-toxic polysaccharide that is extensively studied as a biocompatible vector for gene and drug delivery. However, the fabrication of chitosan nanoparticle (CNP) is usually encountered with a wide size distribution and poor particle stability, which unfortunately limits their role for certain biological applications. This study reports the synthesis and characterisation of CNPs under optimised conditions. The CNPs were synthesised via ionic gelation process utilizing tripolyphosphate (TPP) as a cross-linking agent. The particle size and morphology of samples were subsequently evaluated using dynamic light scattering (DLS), electron microscopy and Fourier-transform infrared spectroscopy (FTIR). Findings arising from this study showed the optimised nanoparticles exhibited spherical shaped CNPs with a size range from 4 to 25nm which lays the foundation for further applications.
Recombinant expression and purification of adenocarcinoma GPR161 receptor
Kasym Kasenovich Mukanov, Zhansaya Batyrbekkyzy Adish, Kanatbek Naizabekovich Mukantayev, Kanat Akhmetovich Tursunov, Zhuldyz Kydyrbekkyzy Kairova, Guldarigash Kuanyshovna Kaukabayeva, Arman Tabylovich Kulyyassov, Pavel Viktorovich Tarlykov
APJMBB 27 (4): 85-95
Article DOI: https://doi.org/10.35118/apjmbb.2019.027.4.10
Click here to download [PDF]
Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer and very few therapeutic options are currently available for its treatment. Interestingly, G-protein coupled receptor 161 (GPR161) is expressed in TNBC cells and can activate the mammalian target of the rapamycin complex 1 signaling pathway. GPR161 and Ras GTPase-activating-like protein, a protein involved in intracellular signaling, proliferation, and cellular adhesion, have been shown to genetically interact in human breast cancer cells. Targeting of GPR161 by monoclonal antibodies may therefore be a strategy to develop diagnostics and therapeutics for TNBC. Thus, to obtain such monoclonal antibodies, we synthesized the GPR161 gene de novo, cloned it into the pET32 expression plasmid, and used the recombinant plasmid to transform the competent BL21 (DE3) strain of Escherichia coli. The recombinant GPR161 gene was designed to contain an N-terminal thioredoxin tag, a thrombin site, the GPR161 sequence, and a C-terminal hexa-histidine tag to facilitate purification by metal-affinity chromatography. Following purification of the recombinant GPR161 (rGPR161) protein using a HisTrap column, we characterized the protein by Western blotting and mass spectrometry. The rGPR161 protein had a molecular mass of ~49 kDa and its identity as rGPR161 was confirmed by mass spectrometry data using the SwissProt database and the Mascot program. Future studies will involve the development of monoclonal antibodies using rGPR161 as the immunogen.

 

About MSMBB

We are a non-profit organisation that was established in 1988 to promote molecular biology and biotechnology.

Stay Connected on:

    

Next Event

3rd International Conference on Molecular Biology & Biotechnology

in conjunction with the 26th  Scientific Meeting of the Malaysian Society of Molecular Biology & Biotechnology (MSMBB) & UCSI University's 2nd Applied Science Symposium

Organized by MSMBB & UCSI University

FOSTERING INNOVATION THROUGH RESEARCH

24-25th April 2019, UCSI University Kuala Lumpur

Visit http://bit.ly/3rdICMBB 

 

 

Contacts Us

For general information about MSMBB, including registration, please contact us at:

  Department of Parasitology,
Faculty of Medicine,
University of Malaya,
50603 Kuala Lumpur,
Malaysia.
  This email address is being protected from spambots. You need JavaScript enabled to view it.
  +603 - 7967 4749
  +603 - 7967 4749