This study was conducted to detect the presence of chicken and porcine DNA in meatballs using mitochondria DNA (mtDNA) of cytochrome b (cyt b) and nuclear DNA (nDNA) short interspersed nuclear element (SINE) species-specific primers, respectively. While, the mtDNA primers targeted transfer RNA-ATP8 (tRNA-ATP8) gene was used for 1 and 5% (w/w) chicken meatball spiked with commercial porcine blood plasm. Chicken meatballs spiked with 1% and 5% (v/w) fresh and commercial porcine blood plasma, respectively were prepared and heat-treated using five (n = 5) cooking methods: boiling, pan-frying, roasting, microwaving and autoclaving. Two pairs of mtDNA and nDNA primers used, produced 129 and 161 bp amplicons, respectively. Whereas, tRNA-ATP8 primers produced 212 bp of amplicon. Electrophoresis analysis showed positive results for porcine DNA at 1% and 5% (w/w or v/v) for all of the different cooking techniques, either for fresh or commercial blood plasma using SINE primers but not for tRNA-ATP8 primers. The present study has highlighted the useful of species-specific primers of SINE primers in PCR analysis for detecting porcine DNA blood plasma in heat-treated chicken meatballs.
Haemosporidians infect a wide diversity of bat genera and species, yet little is known about their transmission cycles or epidemiology. Though several recent studies have focused on the genus Hepatocystis, an Old World parasite primarily infecting bats, monkeys, and squirrels, this group is still understudied with little known about its transmission and molecular ecology. These parasites lack an asexual erythrocytic stage, making them unique from the Plasmodium vertebrate life cycle. In this study, we detected a prevalence of 31% of Hepatocystis in short-nosed fruit bats (Cynopterus brachyotis) in Singapore. Phylogenetic reconstruction with a partial cytochrome b sequence revealed a monophyletic group of Hepatocystis from C. brachyotis in Malaysia, Singapore, and Thailand. There was no relationship with infection and bat age, sex, location, body condition or monsoon season. The absence of this parasite in the five other bat species sampled in Singapore indicates this Hepatocystis species may be host restricted.
Determination of feline meat in food products is an important issue for social, health, economic and religious concern. Hence this paper documented the application of species specific polymerase chain reaction-restriction fragment length polymorphism (SP-PCR-RFLP) assay targeting a short-fragments (69 bp) of mitochondrial cytochrome b (cytb) gene to screen feline meat in commercial meat products using lab-on-a-chip. The SP-PCR assay proved its specificity theoretically and experimentally while testing with different common animal, aquatic and plant species of DNA. The feline specific (69 bp, 43- and 26-bp) characteristic molecular DNA pattern was observed by SP-PCR and RFLP analysis. For assay performance, it was tested in three different types of commercial dummy meat products such as frankfurters, nuggets and meatballs and digested with AluI-restriction enzyme. The highest sensitivity of the assay using lab-on-a-chip was as low as 0.1 pg or 0.01 % (w/w) in commercial dummy meat products. We have also applied this assay to screen three important commercial meat products of six different brand from six supermarket chains located at three different states of Malaysia. Thus total 378 samples were tested to validate the specificity, sensitivity, stability of the assay and utilization of it for commercial meat product screening.
DNA Barcoding, primarily focusing on cytochrome coxidase subunit I (COI) gene has been appraised as an effective tool for species identification. Nonetheless, species identification based on molecular approach is essential for discrimination of look-alike species. In this study, we focused on the marine fishes Family Nemipteridae, one of the commercially important group distributed within the surrounding seas of Malaysia. Some of the samples were collected during the National Demersal Trawl Survey in the Exclusive Economic Zone of East Coast Peninsular Malaysia by the Department of Fishery Malaysia. A 652bp region of COI was sequenced for 74 individuals from nine putative species. Additional 34 COIsequences from GenBank were also included in this study making the total number of samples analysed to 108 individuals. The averageKimura 2-parameter (K2P) nucleotide divergence was 0.34% among individuals within species and 6.97% within genera. All putative species formed monophyletic clades in both Neighbour-joining (NJ) and Maximum-likelihood (ML) trees. However, there was a potential misidentification in specimen identified as Nemipterus tambuloides,as the specimen did not group with their own taxa. It was genetically grouped in Nemipterus thosaporni clade. This study supports the effectiveness of COIgene in species discrimination of Family Nemipteridae.
This study was conducted to investigate the sensitivity and detection of porcine DNA in raw materials, ingredients and finished bakery products by polymerase chain reaction (PCR) - southern hybridization on chip analysis. A total of 20 samples (n=20* 3) with three replicates for each samples were obtained from a bakery factory located in Bangi, Selangor from January to December 2012. The sensitivity level of PCR-southern hybridization on chip was 0.001 ng. The species-specific oligonucleotide primers used in PCR-southern hybridization were targeted on the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene sequence, namely cty b biotin-labeled oligonucleotide primers. The amplicon from PCR amplification was 276 bp in size. None of the raw materials, ingredients and finished bakery product samples was positive towards porcine DNA, except for the positive control. The results in the present study demonstrated that the PCR- southern-hybridization technique on the gene chip (OliproTM Porcine gene chip) is a sensitive tool for monitoring the porcine component in highly processed ingredients and finished bakery products.
The need to detect genetic variation has fueled the development of novel marker systems in fisheries biology. In this study, a simple, fast and cost effective method was used to differentiate between species of freshwater fishes focusing on Malaysian freshwater fishes by employing
Restriction Fragment Length Polymorphisms (RFLPs) analysis of a 470-bp cytochrome b mtDNA segment. RFLP analysis using six restriction enzymes (AluI, BamHI, BsuRI, Csp61, HpaII and SalI) found variations in the digestion profile among most of the fish samples analyzed. Diagnostic digestion profiles were observed among the Hampala fishes, especially between H. macrolepidota and the other Hampala species/forms (using BsuRI and Csp61). Diagnostic digestion profiles were also detected between H.
bimaculata Type A and Type B (using AluI, BamHI, BsuRI and SalI), supporting their status as distinct species. Additionally, unique digestion profiles were observed in other species such as Leptobarbus hosii (Csp61), Osteocheilus hasseltii (Csp61), Osteocheilus sp. (Csp61), Puntioplites bulu (Csp61), Puntius bramoides (AluI), P. sealei (AluI) and Helostoma temmincki (AluI and Csp61), which can be used as genetic markers for discriminating these species. Overall, the RFLP analysis of the cytochrome
b mtDNA segment has proven to be a considerably effective, fast and non-expensive technique to discriminate among several freshwater fish species in Malaysia.
A technique to isolate DNA from ghee was developed for the authentication of beef fat product. The method was based on pre-mixed ghee with phosphate buffer solution (PBS) prior to DNA extraction using Epicentre extraction method. The recovery of beef DNA was then analysed by polymerase chain reaction (PCR) using beef species-specific oligonucleotide primers which targeted the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene. The amplicon was 274 bp in size. The developed ghee extraction method offers a high yield of DNA providing 100 ng per μl and useful for validating beef fat product.
Chrysomya bezziana is an obligate, myiasis-causing fly in humans and warm-blooded animals throughout the tropical and subtropical Old World. We report a case of cutaneous myiasis due to C. bezziana in a dog from Guangxi province in China. A total of 35 maggots were removed from the lesions. Direct sequencing of the mitochondrial cytochrome b gene showed that the specimen belonged to haplotype CB_bezz02, which was previously reported in Malaysia and the Gulf region. This paper also reviews reported cases of screwworm myiasis from humans and animals in China. Geographical records indicate that the distribution of C. bezziana is expanding, suggesting that an integrated pest management control should be taken into consideration in China.
A method of PCR-restriction fragment length polymorphism (RFLP) has been utilized to differentiate the mitochondrial genes of pork and wild boar meat (Sus scrofa). The amplification PCR products of 359 bp and 531 bp were successfully amplified from the cyt b gene of these two meats. The amplification product of pork and wild boar using mt-12S rRNA gene successfully produced a single band with molecular size of 456 bp. Three restriction endonucleases (AluI, HindIII and BsaJI) were used to restrict the amplification products of the mitochondrial genes. The restriction enzymes of AluI and BsaJI were identified as potential restriction endonucleases to differentiate those meats. HindIII enzyme was unable to restrict the PCR product of both meats. The genetic differences within the cyt b gene among the two meats were successfully confirmed by PCR-RFLP analysis.
Slow lorises (Nycticebus) consist of eight species native to Southeast Asia while three species are recognised in
Malaysia - N. coucang, N. menagensis and N. kayan. This study reports on the rediscovery of the subspecies N. coucang
insularis Robinson, 1917 in Tioman Island and the genetic assessment of its mitochondrial DNA variation. Morphological
measurements conform the specimen as the putative N. coucang but with distinct colour and markings. Two mitochondrial
DNA segments (cytochrome b and control region) were produced from the subspecies representing their first registered
sequences in GenBank. Genetically, the subspecies showed 99% of nucleotide similarity to N. coucang species type for
both the DNA segments and constitute its own unique haplotype. Phylogenetic trees constructed using three methods
(neighbour joining, maximum likelihood and Bayesian inference) showed two major groups within Nycticebus; the
basal group was formed by N. pygmaeus while the second group consisted of the remaining Nycticebus species. The
phylogenetic position of the subspecies, however, remains unresolved due to the observed mixing between N. coucang and
N. bengalensis. Several reasons could lead to this condition including the lack of well documented voucher specimens and
the short DNA fragments used. In addition, the possibility of hybridisation event between N. coucang and N. bengalensis
could not be excluded as a possible explanation since both species occur sympatrically at the Isthmus of Kra region
until the Thailand-Malaysia border. The rediscovery of this subspecies displays the unique faunal diversity that justifies
the importance of Tioman Island as a protected area.
Family Scolopacidae includes the sandpipers, shanks, snipes, godwits and curlews. Systematic classifications of shorebirds
at the higher level have been successfully resolved. Nevertheless, the phylogeny of shorebirds in the familial level is still
poorly understood. Thus, this phylogenetic study on Scolopacidae was conducted upon the framework provided by the first
sequence-based species-level phylogeny within the shorebirds to determine the phylogenetic relationships among family
members of Scolopacidae in West Borneo, Sarawak using combined gene markers, mtDNA Cytochrome Oxidise I (COI)
and nucDNA Recombinant Activating Gene 1 (RAG1). A total of 1,342 base pair (bp) were inferred from both COI and RAG1
gene from 45 sequences constituted of 15 species Scolopacidae sampled from Sarawak namely Xenus cinereus, Actitis
hypoleucos, Tringa totanus, Tringa glareola, Tringa stagnatilis, Heteroscelus brevipes, Calidris alba, Calidris ruficollis,
Calidris ferruginea, Calidris tenuirostris, Calidris alpina, Gallinago stenura, Gallinago megala, Numenius arquata, and
Numenius phaeopus. The phylogenetic tree was constructed with Charadrius mongulus derived as an outgroup. The
Bayesian Inference (BI) tree constructed supported grouping of species into several lineages of Numeniinae, Calidrinae,
Scolopacinae and Tringinae. The groupings of species into several lineages correlate with morphological features that
contribute to their adaptation and ability of the species to fit to their ecosystems.
A Mo -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo (10 mM), the bacterium reduced Mo to form molybdenum blue. Approximately 27% of Mo added to the medium was reduced after 28 h of cultivation. The reduction of Mo with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo reduction. NADH and N,N,N',N' -tetramethyl-p-phenylenediamine served as electron donors for Mo reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo reduction. Both ferric and stannous ions strongly enhanced the activity of Mo reduction by NADH.
Sphingobium sp. strain SYK-6 is able to degrade various lignin-derived biaryls, including a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA). In SYK-6 cells, the alcohol group of the B-ring side chain of DCA is initially oxidized to the carboxyl group to generate 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylic acid (DCA-C). Next, the alcohol group of the A-ring side chain of DCA-C is oxidized to the carboxyl group, and then the resulting metabolite is catabolized through vanillin and 5-formylferulate. In this study, the genes involved in the conversion of DCA-C were identified and characterized. The DCA-C oxidation activities in SYK-6 were enhanced in the presence of flavin adenine dinucleotide and an artificial electron acceptor and were induced ca. 1.6-fold when the cells were grown with DCA. Based on these observations, SLG_09480 (phcC) and SLG_09500 (phcD), encoding glucose-methanol-choline oxidoreductase family proteins, were presumed to encode DCA-C oxidases. Analyses of phcC and phcD mutants indicated that PhcC and PhcD are essential for the conversion of (+)-DCA-C and (-)-DCA-C, respectively. When phcC and phcD were expressed in SYK-6 and Escherichia coli, the gene products were mainly observed in their membrane fractions. The membrane fractions of E. coli that expressed phcC and phcD catalyzed the specific conversion of DCA-C into the corresponding carboxyl derivatives. In the oxidation of DCA-C, PhcC and PhcD effectively utilized ubiquinone derivatives as electron acceptors. Furthermore, the transcription of a putative cytochrome c gene was significantly induced in SYK-6 grown with DCA. The DCA-C oxidation catalyzed by membrane-associated PhcC and PhcD appears to be coupled to the respiratory chain.
Zerumbone (ZER) isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl- β -cyclodextrin (HP β CD) to enhance ZER's solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HP β CD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HP β CD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HP β CD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.
Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system.
Three species of flying fox (Pteropus hypomelanus, P. vampyrus, and P. lylei) from Malaysia and Vietnam were screened for apicomplexan parasites by thin blood smears and polymerase chain reaction. Only 1 of 16 bats sampled from 3 localities in southeast Asia was found to be infected (P. hypomelanus from Pulau Pangkor, Malaysia). We observed micro- and macrogametocytes, with morphology consistent with Hepatocystis sp. parasites, using light microscopy. Phylogenetic analysis of the cytochrome b gene showed that the parasite from P. hypomelanus groups with 2 published sequences from Hepatocystis spp., including one from Cynopterus brachyotis, another fruit bat in the Pteropodidae.
Food fraud is a global problem raising increased concerns during the past decades and food authenticity is now a burning issue. Beef, buffalo, chicken, duck, goat, sheep, and pork are heavily consumed meats bearing nutritional, economic and cultural/religious importance and are often found to be adulterated in raw and processed states. To authenticate these species, we developed and validated a highly specific multiplex (heptaplex) PCR assay targeting short length amplicons (73-263 bp) using seven pairs of species-specific primer sets targeting mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes. Specificity checking (in silico and in vitro) against 25 non-target species revealed no cross-species amplification. The developed multiplex assay was validated with various adulterated and heat-treated (boiled, microwaved and autoclaved) meatball products and were found to show high sensitivity and stability under all processing conditions. The assay was sensitive enough to detect 0.01-0.005 ng of DNA from raw meat and 0.5% (w/w) adulterated meat in mixed matrices. A market survey revealed mislabelling of 95% beef and 15% chicken products while pork products were found pure. Given some advantageous features including short sizes of amplicons, exceptional stability and superior sensitivity, the developed assay could be conveniently used for discriminatory detection of target species with a variety of raw meat as well as processed meat products undergoing extreme processing treatments.
Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.
OBJECTIVES: To investigate the cytotoxic effect of anastrozole on breast (MCF7), liver hepatocellular (HepG2), and prostate (PC3) cancer cells. Methods: This is a prospective study. Anastrozole's mechanism of apoptosis in living cells was also determined by high content screening (HCS) assay. Methylthiazol tetrazolium (MTT) assay was carried out at the Centre of Biotechnology Research's, Al-Nahrain University, Baghdad, Iraq between July 2015 and October 2015. The HCS assay was performed at the Centre for Natural Product Research and Drug Discovery, Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia between November 2015 and February 2016. Results: The most significant cytotoxic effect of anastrozole towards 3 cancer cell lines was obtained when its concentration was 400 µg/mL. The MCF7 cells were more sensitive to anastrozole compared with the HepG2 and PC-3 cells. There was a significant increase in membrane permeability, cytochrome c and nuclear intensity when anastrozole (200 µg/mL) was used compared with doxorubicin (20 µg/mL) as a standard. Also, there was a significant decrease in cell viability and mitochondrial membrane permeability when anastrozole (200 µg/mL) was used compared with positive control. Conclusion: Anastrozole showed cytotoxic effects against the MCF7, HepG2, and PC3 cell lines as determined in-vitro by the MTT assay. The HCS technique also showed toxic effect towards MCF7. It is evident that anastrozole inhibits the aromatase enzyme preventing the aromatization mechanism; however, it has a toxic effect.
This study was conducted to identify and to compare the mitochondrial morphological alterations in livers of rats treated with various doses of diclofenac and ibuprofen. Hundred and forty-four male Sprague Dawley rats were dosed with 3, 5 and 10 mg kg(-1) diclofenac and ibuprofen in saline via intraperitoneal injection for 15 days. The control group was administered with saline in a similar manner. Four rats were euthanised every 3 days until day 15. While 200 mg kg(-1) diclofenac and ibuprofen-treated rats (n = 4) were euthanized 10 h posttreatment. The livers were removed, cleaned and a section across the right lobe was taken and fixed in 4% (v/v) glutaraldehyde for electron microscopy analysis and the remaining samples were kept at -80 degrees C for Western blot analysis. Five milligram per kilogram and 10 mg kg(-1) diclofenac-administered rats for 15 days revealed the presence of enlarged mitochondria, irregular and ruptured mitochondrial membranes. While rats administered with 10 mg kg(-1) ibuprofen also showed the presence of mitochondria with irregular membrane structure and ruptured membranes. Western blotting analysis of mitochondrial fractions revealed the expression of cytochrome c in all samples and complete absence of cytochrome c expression in the cytosolic fraction of all samples after day 15. Analysis in 200 mg kg(-1) diclofenac and ibuprofen-treated groups, revealed expression of cytochrome c in both mitochondrial and cytosolic fractions. This observation indicates that both diclofenac and ibuprofen may alter the morphology of mitochondria, leading to cytochrome c release into the cytosol. Further studies needs to be conducted to investigate on the activity of the mitochondria following both treatments.