Phyla nodiflora L. has been used as medicinal remedies for various ailments due to its antioxidant, anti-inflammatory, anti-bacterial, anti-tumor activity. Previously, we found that the plant extracts induced DNA fragmentation in MCF-7. This study was to investigate the modes of action of P. nodiflora in inhibiting breast cancer cells using leaf ethyl acetate (EA leaf), stem ethyl acetate (EA stem) and stem methanol (Met stem) extracts. The MTT assay showed that the anti-proliferative effects of P. nodiflora extracts were selective towards MCF-7 with a minimal effect on MCF10A. Morphological changes such as cell shrinkage and nuclear condensation were observed in treated cells. We found that induction of apoptosis by EA leaf and EA stem was mitochondrial-dependent while loss of mitochondrial membrane potential was not found in Met stem-treated cells. In addition, the expression levels of AIFM1, CASP9, CFLAR, and IGF1R were altered after treatment. Decreased BCL-2 expression was found in treated cells while BAX and caspases' expression was upregulated or maintained. All extracts caused perturbation of cell cycle at S phase by dysregulating the expression of cell cycle regulators such as CDKs and cyclins. Our findings indicate that P. nodiflora inhibits MCF-7 cells by inducing apoptosis and perturbing cell cycle.
The relationship between diabetes and endothelial dysfunction remains unclear, particularly the association with pathological activation of calpain, an intracellular cysteine protease. Here, we used human induced pluripotent stem cells-derived endothelial cells (iPSC-ECs) to investigate the effects of diabetes on vascular health. Our results indicate that iPSC-ECs exposed to hyperglycemia had impaired autophagy, increased mitochondria fragmentation, and was associated with increased calpain activity. In addition, hyperglycemic iPSC-ECs had increased susceptibility to cell death when subjected to a secondary insult-simulated ischemia-reperfusion injury (sIRI). Importantly, calpain inhibition restored autophagy and reduced mitochondrial fragmentation, concurrent with maintenance of ATP production, normalized reactive oxygen species levels and reduced susceptibility to sIRI. Using a human iPSC model of diabetic endotheliopathy, we demonstrated that restoration of autophagy and prevention of mitochondrial fragmentation via calpain inhibition improves vascular integrity. Our human iPSC-EC model thus represents a valuable platform to explore biological mechanisms and new treatments for diabetes-induced endothelial dysfunction.
Holothuria (Mertensiothuria) leucospilota (Brandt 1835), white threads fish or locally known as bat puntil is currently considered as the most abundant sea cucumber species in Malaysia. This study aimed to generate the genetic profile of H. leucospilota from Malaysia and then to determine the phylogenetic relationship between H. leucospilota and other members of genus Holothuria using partial sequences of cytochrome c oxidase I (COI) mitochondrial DNA (mtDNA) gene. In this study, specimens of H. leucospilota were collected from Intan Besar Island, Langkawi, Kedah Darul Aman in the west coast of Peninsular Malaysia. Three main methods namely neighbour joining, maximum parsimony and maximum likelihood were used for the phylogenetic tree reconstruction. Tree topologies showed that H. leucospilota has its own monophyletic clade clearly distinct from the other species. The pairwise genetic distance calculated further supported these findings. In addition, the results also should that the COI mtDNA gene is capable to unravel the phylogenetic relationship of H. leucospilota.
Cyanide is highly toxic to the living organisms as it inhibits respiration system in the cell mitochondria. Cyanide is commonly used in gold extraction process and its discharge into the environment not only causes pollution but it also brings harm to the surrounding population. Chemical treatment is expensive and the use of hazardous compound can exacerbate the problem. Biodegradation offers cheap and safe alternative as it overcomes the problems faced by chemical treatment. In this study, indigenous bacteria from mining wastewater were isolated. Cyanide degradation was done via shake flask method. A bacterium, designated W2 was found able to grow in the mining wastewater. 16S rRNA analysis identified the strain as Pseudomonas pseudoalcaligenes which could tolerate up to 39 mg/L cyanide concentration and growth was depleted at 52 mg/L. 60% cyanide degradation was achieved in wastewater containing medium. End-product analysis from high performance liquid chromatography (HPLC) detected formamide implicating the role of cyanide hydratase in cyanide degradation. It can be concluded that P. pseudoalcaligenes is capable of biodegrading cyanide and its potential in wastewater treatment containing cyanide is feasible.
This study aimed to resolve the taxonomic status of a morphologically undetermined sea cucumber species of order Apodida
from Malaysia (GenBank accession no.: FJ223867) using partial 16S mitochondrial rRNA gene sequences and subsequently
to determine the validity of morphological taxonomy of Holothuria species into its current subgenera. The undetermined
species clustered with all taxa of Holothuria in previous study. Phylogenetic analyses using maximum parsimony and
Bayesian methods suggest that the undetermined species was genetically closer to Holothuria (Lessonothuria) pardalis and
Holothuria (Acanthotrapeza) coluber; and its position in both phylogenetic trees further suggests its status as a Holothuria
taxon. Subgenera of Holothuria, Merthensiothuria and Metriatyla are monophyletic with strong bootstrap supports and
posterior probabilities of clades, thus strengthening their morphological taxonomies. Nonetheless, the non-monophyly of
subgenera of Halodeima, Microthele and Platyperona suggests a requirement for their taxonomic revisions using integrative
taxonomy. The status of Holothuria (Halodeima) edulis subgroups in the maximum parsimony and Bayesian trees is
indistinct and further taxonomic revisions are necessary. In terms of sister relationship, both phylogenetic trees suggest
that subgenus Holothuria is a sister taxon of subgenus Roweothuria while the other sister relationships were unclear due
to the undetermined species, paraphyly and polyphyly of a number of subgenera. Further studies with more specimens of
genus Holothuria from broader geographical locations and various mtDNA genes along with morphological approaches
may facilitate to provide better insights into the molecular phylogeny of subgenera of Holothuria.
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.
The rationale of designing compounds containing a (3,4,5-trimethoxybenzyloxy) phenyl moiety is largely due to its potential antioxidant and cytotoxic activities. A previous study focused on its antioxidant mechanism, whereas in this study, we investigated the cytotoxicity of a series of 28 analogues and the mechanism of apoptosis of the most cytotoxic compound against wild-type (HCT-116) and p53 mutant (HT-29) colorectal cancer cell lines. The series of analogues comprise of different families, namely hydrazone, oxadiazole, thiosemicarbazides and triazoles. In the initial cytotoxicity screening, N-(3,4,5-trimethoxybenzylidene)-4-(3,4,5-trimethoxybenzyloxy) benzohydrazide, henceforth known as, P5H, was found to be most cytotoxic against human colorectal cancer cell lines (IC50 for HCT-116 = 11.79 μM and HT-29 = 18.52 μM). Additionally, P5H was found to have some degree of selectivity towards cancer cells compared to normal human colon cells (CCD-112 CoN). Subsequent investigation had brought insight on P5H ability to induce apoptosis in both HCT-116 and HT-29 cell lines. Cell cycle analysis showed both cell lines were arrested at the G2/M phase upon treatment. Our study concluded that P5H induced the death receptor, DR5 in HCT-116 and mitochondria-mediated apoptosis pathway in HT-29. Therefore, P5H may be a promising candidate as a chemotherapy agent against colon cancer. Graphical abstract The apoptotic pathways induced in HT-29 and HCT-116 cells upon P5H treatment.
This article contains data of the sequence variation in the mitochondrial DNA D-loop region of the Malayan gaur (Bos gaurus hubbacki), locally known as the seladang, from two captive centers. Thirty fecal samples of Malayan gaur were collected from Jenderak Selatan Wildlife Conservation Center (Pahang) and the Sungkai Wildlife Reserve (Perak) for DNA extraction and amplification with polymerase chain reactions. DNA sequences were then analyzed using neighbor joining (NJ) and maximum parsimony (MP) methods. Based on the 652 base pairs obtained, we found seven variable characters with a value of 1%. The genetic distance between the two captive centers was 0.001. Haplotype analyses detected only four haplotypes between these two captive centers. Both NJ and MP trees demonstrate that all individuals in the Jenderak and Sungkai captive centers are in the same clade. Genetic variation of the Malayan gaur in these centers is considered low, possibly because individuals share the same common parent. This sequence variation data are of paramount importance for designing a proper breeding and management program of the Malayan gaur in the future.
Mitochondria are best known for their role in energy production, and they are the only mammalian organelles that contain their own genomes. The mitochondrial genome mutation rate is reported to be 10-17 times higher compared to nuclear genomes as a result of oxidative damage caused by reactive oxygen species during oxidative phosphorylation. Pathogenic mitochondrial DNA mutations result in mitochondrial DNA disorders, which are among the most common inherited human diseases. Interventions of mitochondrial DNA disorders involve either the transfer of viable isolated mitochondria to recipient cells or genetically modifying the mitochondrial genome to improve therapeutic outcome. This review outlines the common mitochondrial DNA disorders and the key advances in the past decade necessary to improve the current knowledge on mitochondrial disease intervention. Although it is now 31 years since the first description of patients with pathogenic mitochondrial DNA was reported, the treatment for mitochondrial disease is often inadequate and mostly palliative. Advancements in diagnostic technology improved the molecular diagnosis of previously unresolved cases, and they provide new insight into the pathogenesis and genetic changes in mitochondrial DNA diseases.
The southern lesser pomfret (Pampus minor) is an economically important fish, and its numbers are declining because of overfishing and environmental pollution. In addition, owing to the similarities of its external morphological characteristics to other species in the genus Pampus, it is often mistaken for grey pomfret (P. cinereus) or silver pomfret (P. argenteus) juveniles. In this study, the genetic diversity and structure of 264 P. minor individuals from 11 populations in China and Malaysia coastal waters were evaluated for the first time, to the best of our knowledge, using mitochondrial cytochrome b fragments. The results showed that P. minor had moderate haplotype diversity and low nucleotide diversity. Furthermore, two divergent lineages were detected within the populations, but the phylogenetic structure corresponded imperfectly with geographical location; thus, the populations may have diverged in different glacial refugia during the Pleistocene low sea levels. Analysis of molecular variation (AMOVA) showed that genetic variation originated primarily from individuals within the population. Pairwise FST results showed significant differentiation between the Chinese and Malaysian populations. Except for the Xiamen population, which was classified as a marginal population, the genetic differentiation among the other Chinese populations was not significant. During the Late Pleistocene, P. minor experienced a population expansion event starting from the South China Sea refugium that expanded outward, and derivative populations quickly occupied and adapted to the new habitat. The results of this study will provide genetic information for the scientific conservation and management of P. minor resources.
This study investigated the impact of prolonged nicotine administration on myocardial susceptibility to ischaemia-reperfusion (I/R) injury in a rat model and determined whether nicotine affects mitochondrial reactive oxygen species (ROS) production and permeability transition in rat hearts. Sprague-Dawley rats were administered 0.6 or 1.2 mg/kg nicotine for 28 days, and their hearts were isolated at end-point for assessment of myocardial susceptibility to I/R injury ex vivo. Rat heart mitochondria were also isolated from a subset of rats for analysis of mitochondrial ROS production and permeability transition. Compared to the vehicle controls, rat hearts isolated from nicotine-administered rats exhibited poorer left ventricular function that worsened over the course of I/R. Coronary flow rate was also severely impaired in the nicotine groups at baseline and this worsened after I/R. Nicotine administration significantly increased mitochondrial ROS production and permeability transition relative to the vehicle controls. Interestingly, pre-incubation of isolated mitochondria with ROS scavengers (superoxide dismutase and mitoTEMPO) significantly abolished nicotine-induced increase in mitochondria permeability transition in isolated rat heart mitochondria. Overall, our data showed that prolonged nicotine administration enhances myocardial susceptibility to I/R injury in rats and this is associated with mitochondrial ROS-driven increase in mitochondrial permeability transition.
Invasive apple snails, Pomacea canaliculata and P. maculata, have a widespread distribution globally and are regarded as devastating pests of agricultural wetlands. The two species are morphologically similar, which hinders species identification via morphological approaches and species-specific management efforts. Advances in molecular genetics may contribute effective diagnostic tools to potentially resolve morphological ambiguity. DNA barcoding has revolutionized the field of taxonomy by providing an alternative, simple approach for species discrimination, where short sections of DNA, the cytochrome c oxidase subunit I (COI) gene in particular, are used as 'barcodes' to delineate species boundaries. In our study, we aimed to assess the effectiveness of two mitochondrial markers, the COI and 16S ribosomal deoxyribonucleic acid (16S rDNA) markers for DNA barcoding of P. canaliculata and P. maculata. The COI and 16S rDNA sequences of 40 Pomacea specimens collected from six localities in Peninsular Malaysia were analyzed to assess their barcoding performance using phylogenetic methods and distance-based assessments. The results confirmed both markers were suitable for barcoding P. canaliculata and P. maculata. The phylogenies of the COI and 16S rDNA markers demonstrated species-specific monophyly and were largely congruent with the exception of one individual. The COI marker exhibited a larger barcoding gap (6.06-6.58%) than the 16S rDNA marker (1.54%); however, the magnitude of barcoding gap generated within the barcoding region of the 16S rDNA marker (12-fold) was bigger than the COI counterpart (approximately 9-fold). Both markers were generally successful in identifying P. canaliculata and P. maculata in the similarity-based DNA identifications. The COI + 16S rDNA concatenated dataset successfully recovered monophylies of P. canaliculata and P. maculata but concatenation did not improve individual datasets in distance-based analyses. Overall, although both markers were successful for the identification of apple snails, the COI molecular marker is a better barcoding marker and could be utilized in various population genetic studies of P. canaliculata and P. maculata.
Cisplatin is the first-line chemotherapeutic agent for the treatment of oral squamous cell carcinoma (OSCC). However, the intrinsic or acquired resistance against cisplatin remains a major obstacle to treatment efficacy in OSCC. Recently, mitochondrial DNA (mtDNA) alterations have been reported in a variety of cancers. However, the role of mtDNA alterations in OSCC has not been comprehensively studied. In this study, we evaluated the correlation between mtDNA alterations (mtDNA content, point mutations, large-scale deletions, and methylation status) and cisplatin sensitivity using two OSCC cell lines, namely SAS and H103, and stem cell-like tumour spheres derived from SAS. By microarray analysis, we found that the tumour spheres profited from aberrant lipid and glucose metabolism and became resistant to cisplatin. By qPCR analysis, we found that the cells with less mtDNA were less responsive to cisplatin (H103 and the tumour spheres). Based on the findings, we theorised that the metabolic changes in the tumour spheres probably resulted in mtDNA depletion, as the cells suppressed mitochondrial respiration and switched to an alternative mode of energy production, i.e. glycolysis. Then, to ascertain the origin of the variation in mtDNA content, we used MinION, a nanopore sequencer, to sequence the mitochondrial genomes of H103, SAS, and the tumour spheres. We found that the lower cisplatin sensitivity of H103 could have been caused by a constellation of genetic and epigenetic changes in its mitochondrial genome. Future work may look into how changes in mtDNA translate into an impact on cell function and therefore cisplatin response.
Mitochondria have multiple functions, including synthesis of adenine triphosphate, production of reactive oxygen species, calcium signalling, thermogenesis and apoptosis. Mitochondria have a significant contribution in regulating the various physiological aspects of reproductive function, from spermatogenesis up to fertilisation. Mitochondrial functionality and intact mitochondrial membrane potential are a pre-requisite for sperm motility, hyperactivation, capacitation, acrosin activity, acrosome reaction and DNA integrity. Optimal mitochondrial activity is therefore crucial for human sperm function and semen quality. However, the precise role of mitochondria in spermatozoa remains to be fully explored. Defects in sperm mitochondrial function severely impair the maintenance of energy production required for sperm motility and may be an underlying cause of asthenozoospermia. Sperm mtDNA is susceptible to oxidative damage and mutations that could compromise sperm function leading to infertility. Males with abnormal semen parameters have increased mtDNA copy number and reduced mtDNA integrity. This review discusses the role of mitochondria in sperm function, along with the causes and impact of its dysfunction on male fertility. Greater understanding of sperm mitochondrial function and its correlation with sperm quality could provide further insights into their contribution in the assessment of the infertile male.
Sarcopenia refers to the involuntary and generalized deterioration of skeletal muscle mass and strength, which may lead to falls, frailty, physical disability, loss of independence, morbidity and mortality. The majority of molecular and cellular changes involved in the degeneration of muscle tissues are mediated by oxidative stress. Therefore, astaxanthin may act as a potential adjunct therapy for sarcopenia owing to its antioxidant activity. The present review examines the effects of astaxanthin on the promotion of skeletal muscle performance and prevention of muscle atrophy and the potential mechanisms underlying these effects. The available evidence till date was retrieved from PubMed and Medline electronic databases. The present review reported the beneficial effects of astaxanthin in preventing muscle degeneration in various animal models of sarcopenia. In humans, the effects of astaxanthin in combination with other antioxidants on muscle health are mixed, wherein positive and negligible effects were reported. Mechanistic studies revealed that astaxanthin promotes muscle health by reducing oxidative stress, myoblast apoptosis and proteolytic pathways while promoting mitochondria regeneration and formation of blood vessels. Thus, astaxanthin is a potential therapeutic agent for sarcopenia but its effects in humans require further validation.
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.
This study was carried out to compare the ultrastructure of fresh, capacitated and acrosome-reacted sperm. The sperm was treated with heparin for capacitation and calcium ionophore for acrosome reaction induction. Sperm samples were then prepared for ultrastructural studies and examined by transmission electron microscopy (TEM). Ultrastructural changes in plasma and acrosomal membranes, shape of the mitochondria and outer dense fibres, in capacitated and acrosome-reacted sperm were evident. The plasma membrane of fresh sperm was loosely fitted around the sperm head and the acrosomal membrane was closely opposed to the nucleus. The plasma and acrosomal membranes of the capacitated sperm were expanded, but disintegrated in the acrosome-reacted sperm. Mitochondria of fresh sperm appeared to be rounded in shape with plasma membrane closely opposed to it and the nine outer dense fibres were almost regular rounded in shape. However, in both capacitated and acrosome-reacted sperm, the mitochondria were almost regular and elongated in shape whilst the outer dense fibres were irregular in shape in the capacitated and acrosome-reacted sperm. There were no noticeable morphological changes found in the axonemal complexes in fresh, capacitated and acrosome-reacted sperm. Ultrastructural studies are able to provide detailed information on sequential events involving numerous physiological changes during fertilization.
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.
Dentatin (DEN), purified from the roots of Clausena excavata Burm f., has poor aqueous solubility that reduces its therapeutic application. The aim of this study was to assess the effects of DEN-HPβCD (hydroxypropyl-β-cyclodextrin) complex as an anticancer agent in HT29 cancer cell line and compare with a crystal DEN in dimethyl sulfoxide (DMSO). The exposure of the cancer cells to DEN or DEN-HPβCD complex leads to cell growth inhibition as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To analyze the mechanism, in which DEN or DEN-HPβCD complex causes the death in human colon HT29 cancer cells, was evaluated by the enzyme-linked immunosorbent assay (ELIZA)-based assays for caspase-3, 8, 9, and reactive oxygen species (ROS). The findings showed that an anti-proliferative effect of DEN or DEN-HPβCD complex were via cell cycle arrest at the G2/M phase and eventually induced apoptosis through both mitochondrial and extrinsic pathways. The down-regulation of poly(ADP-ribose) polymerase (PARP) which leaded to apoptosis upon treatment, was investigated by Western-blotting. Hence, complexation between DEN and HPβCD did not diminish or eliminate the effective properties of DEN as anticancer agent. Therefore, it would be possible to resolve the conventional and current issues associated with the development and commercialization of antineoplastic agents in the future.
The role of mitochondria in tumorigenesis has regained much attention as it could dysregulate cellular energetics, oxidative stress and apoptosis. However, the role of mitochondria in different grade gliomasis still unknown. This study aimed to identify mitochondrial DNA (mtDNA) sequence variations that could possibly affect the mitochondrial functions and also the oxidative stress status. Three different grades of human glioma cell lines and a normal human astrocyte cell line were cultured in-vitro and tested for oxidative stress biomarkers. Relative oxidative stress level, mitochondria activity, and mitochondrial mass were determined by live cell imaging with confocal laser scanning microscope using CM-H2DCFDA, MitoTracker Green, and MitoTracker Orange stains. The entire mitochondrial genome was sequenced using the AffymetrixGeneChip Human Mitochondrial Resequencing Array 2.0. The mitochondrial sequence variations were subjected to phylogenetic haplogroup assessment and pathogenicity of the mutations were predicted using pMUT and PolyPhen2. The Grade II astrocytoma cells showed increased oxidative stress wherea high level of 8-OHdG and oxidative stress indicator were observed. Simultaneously, Grade II and III glioma cells showed relatively poor mitochondria functions and increased number of mutations in the coding region of the mtDNA which could be due to high levels of oxidative stress in these cells. These non-synonymous mtDNA sequence variations were predicted to be pathogenic and could possibly lead to protein dysfunction, leading to oxidative phosphorylation (OXPHOS) impairment, mitochondria dysfunction and could create a vicious cycle of oxidative stress. The Grade IV cells had no missense mutation but preserved intact mitochondria and excellent antioxidant defense mechanisms thus ensuring better survival. In conclusion, Grade II and III glioma cells demonstrated coding region mtDNA mutations, leading to mitochondrial dysfunction and higher oxidative stress.