Hand, foot and mouth disease is mainly caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16), but EV-A71 is also associated with severe neurological complications. Host factors may contribute to the different clinical outcomes of EV-A71 and CV-A16 infections. A neurovirulent EV-A71 strain (EV-A71/UH1) from a fatal case, a non-neurovirulent EV-A71 strain (EV-A71/Sha66) and a CV-A16 strain (CV-A16/22159) from cases of uncomplicated HFMD were used. Replication of the viruses in SK-N-MC (neuronal) and HT-29 (intestinal) cell lines correlated with the severity of clinical disease associated with each virus. EV-A71/UH1 showed the greatest replication in neuronal cells. In HT-29 cells, both EV-A71 strains replicated well, but CV-A16/22159 showed no effective replication. The proteomes of mock and infected SK-N-MC and HT-29 cell lines were compared by 2D-SDS-PAGE. The differentially expressed proteins were identified by MALDI-TOF/TOF analysis. There were 46 and 44 differentially expressed proteins identified from SK-N-MC and HT-29 cells, respectively, categorized under apoptosis, stress, cytoskeletal, energy metabolism proteins and others. Western blot validation showed that EV-A71/UH1 and CV-A16 also differentially induced proteins involved in viral RNA translation and host cell stress responses in neuronal and intestinal cell lines.
This article reports the in vitro degradation and cytotoxicity assessment of Zn-3Mg alloy developed for biodegradable bone implants. The alloy was prepared using casting, and its microstructure was composed of Mg2Zn11 intermetallic phase distributed within a Zn-rich matrix. The degradation assessment was done using potentiodynamic polarization and electrochemical impedance spectrometry. The cell viability and the function of normal human osteoblast cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and alkaline phosphatase extracellular enzyme activity assays. The results showed that the degradation rate of the alloy was slower than those of pure Zn and pure Mg due to the formation of a high polarization resistance oxide film. The alloy was cytocompatible with the normal human osteoblast cells at low concentrations (<0.5 mg/mL), and its alkaline phosphatase activity was superior to pure Mg. This assessment suggests that Zn-3Mg alloy has the potential to be developed as a material for biodegradable bone implants, but the toxicity limit must be carefully observed.
Muscle, a multinucleate syncytium formed by the fusion of mononuclear myoblasts, arises from quiescent progenitors (satellite cells) via activation of muscle-specific transcription factors (MyoD, Myf5, myogenin: MYOG, and MRF4). Subsequent to a decline in Pax7, induction in the expression of MYOG is a hallmark of myoblasts that have entered the differentiation phase following cell cycle withdrawal. It is evident that MYOG function cannot be compensated by any other myogenic regulatory factors (MRFs). Despite a plethora of information available regarding MYOG, the mechanism by which MYOG regulates muscle cell differentiation has not yet been identified. Using an RNA-Seq approach, analysis of MYOG knock-down muscle satellite cells (MSCs) have shown that genes associated with cell cycle and division, DNA replication, and phosphate metabolism are differentially expressed. By constructing an interaction network of differentially expressed genes (DEGs) using GeneMANIA, cadherin-associated protein (CTNNA2) was identified as the main hub gene in the network with highest node degree. Four functional clusters (modules or communities) were identified in the network and the functional enrichment analysis revealed that genes included in these clusters significantly contribute to skeletal muscle development. To confirm this finding, in vitro studies revealed increased expression of CTNNA2 in MSCs on day 12 compared to day 10. Expression of CTNNA2 was decreased in MYOG knock-down cells. However, knocking down CTNNA2, which leads to increased expression of extracellular matrix (ECM) genes (type I collagen α1 and type I collagen α2) along with myostatin (MSTN), was not found significantly affecting the expression of MYOG in C2C12 cells. We therefore propose that MYOG exerts its regulatory effects by acting upstream of CTNNA2, which in turn regulates the differentiation of C2C12 cells via interaction with ECM genes. Taken together, these findings highlight a new mechanism by which MYOG interacts with CTNNA2 in order to promote myoblast differentiation.
Malignant pleural mesothelioma (MPM) is a rare and essentially incurable malignancy most often linked with occupational exposure to asbestos fibres. In common with other malignancies, the development and progression of MPM is associated with extensive dysregulation of cell cycle checkpoint proteins that modulate cell proliferation, apoptosis, DNA repair and senescence.
The present study reports a bioassay-guided isolation of β-caryophyllene from the essential oil of Aquilaria crassna. The structure of β-caryophyllene was confirmed using FT-IR, NMR and MS. The antimicrobial effect of β-caryophyllene was examined using human pathogenic bacterial and fungal strains. Its anti-oxidant properties were evaluated by DPPH and FRAP scavenging assays. The cytotoxicity of β-caryophyllene was tested against seven human cancer cell lines. The corresponding selectivity index was determined by testing its cytotoxicity on normal cells. The effects of β-caryophyllene were studied on a series of in vitro antitumor-promoting assays using colon cancer cells. Results showed that β-caryophyllene demonstrated selective antibacterial activity against S. aureus (MIC 3 ± 1.0 µM) and more pronounced anti-fungal activity than kanamycin. β-Caryophyllene also displayed strong antioxidant effects. Additionally, β-caryophyllene exhibited selective anti-proliferative effects against colorectal cancer cells (IC50 19 µM). The results also showed that β-caryophyllene induces apoptosis via nuclear condensation and fragmentation pathways including disruption of mitochondrial membrane potential. Further, β-caryophyllene demonstrated potent inhibition against clonogenicity, migration, invasion and spheroid formation in colon cancer cells. These results prompt us to state that β-caryophyllene is the active principle responsible for the selective anticancer and antimicrobial activities of A. crassnia. β-Caryophyllene has great potential to be further developed as a promising chemotherapeutic agent against colorectal malignancies.
This unit provides information how to use short interfering RNA (siRNA) for sequence-specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell transfection and transduction, are presented.
Plant mediated synthesis of nanoparticles has been considered as green route and a reliable technique for the synthesis of nanoparticles due to its eco-friendly approach. In this study, we report a simple and eco-friendly approach for the synthesis of silver nanoparticles (AgNPs) using methanolic Momordica cymbalaria fruit extract as reducing agent. The fruit extract of M. cymbalaria exposed to AgNO3 solution showed the change in color from green to light yellow at room temperature within 1h of incubation confirms the synthesis of AgNPs. UV-vis spectra analysis revealed that the synthesized AgNPs had a sharp surface plasmon resonance at around 450 nm, while, the X-ray Diffraction (XRD) patterns confirmed distinctive peaks indices to the crystalline planes of the face centered cubic silver. The Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) analysis results confirmed the presence of spherical shaped AgNPs by a huge disparity in the particle size distribution with an average size of 15.5 nm. The synthesized AgNPs showed strong antibacterial activity against all the tested multidrug resistant human pathogenic bacterial strains and also exhibited highest free radical scavenging activity (74.2%) compared to fruit extract (60.4%). Moreover, both fruit extract and the synthesized AgNPs showed the cytotoxicity towards Rat L6 skeletal muscle cell line at different concentrations, but the highest inhibition percentage was recorded for AgNPs at concentration of 100 μg/ml.
Human pluripotent stem cells (hPSCs) derived from either blastocyst stage embryos (hESCs) or reprogrammed somatic cells (iPSCs) can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as for in vitro modeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulate in vivo conditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs.
Breast cancer is the malignant tumour that developed from cells of the breast and is the first leading cause of cancer death among women worldwide. Surgery, radiotherapy, and chemotherapy are the available treatments for breast cancer, but these were reported to have side effects. Newcastle disease virus (NDV) known as Avian paramyxovirus type-1 (APMV1) belongs to the genus Avulavirus in a family Paramyxoviridae. NDV is shown to be a promising anticancer agent, killing tumour cells while sparing normal cells unharmed. In this study, the oncolytic and cytotoxic activities of NDV AF2240 strain were evaluated on MDA-MB-231, human mammary carcinoma cell line, using MTT assay, and its inhibitory effects were further studied using proliferation and migration assays. Morphological and apoptotic-inducing effects of NDV on MD-MB-231 cells were observed using phase contrast and fluorescence microscopes. Detection of DNA fragmentation was done following terminal deoxyribonucleotide transferase-mediated Br-dUTP nick end labeling staining (TUNEL) assay, which confirmed that the mode of death was through apoptosis and was quantified by flow cytometry. Furthermore, analysis of cellular DNA content demonstrated that the virus caused an increase in the sub-G1 phase (apoptotic peak) of the cell cycle. It appears that NDV AF2240 strain is a potent anticancer agent that induced apoptosis in time-dependent manner.
14-deoxyandrographolide (DA) and 14-deoxy-11,12-didehydroandrographolide (DDA) are two diterpenoids isolated from A. paniculata, a popular folk medicine used as an antihypertensive drug in Malaysia. We have previously reported that DDA exhibited a greater hypotensive effect in anaesthetized rats and a vasorelaxant activity in isolated rat aorta, compared with DA. Their vasorelaxant activities were mediated through the activation of the enzymes, nitric oxide synthase (NOS) and guanylyl cyclase. The present study demonstrated that both DA and DDA stimulated nitric oxide (NO) release from human endothelial cells. DDA compared with DA caused a greater production of NO; this is in line with the finding of the earlier study that the vasorelaxant effect of DDA was more dependent on endothelium than DA.
Crude extracts (methanol) of various parts, viz. the leaves, fruits, roots, stem and trunk bark, of Garcinia atroviridis were screened for antimicrobial, cytotoxic, brine shrimp toxic, antitumour-promoting and antioxidant activities. The crude extracts exhibited predominantly antibacterial activity with the root extract showing the strongest inhibition against the test bacteria at a minimum inhibitory dose (MID) of 15.6 microg/disc. Although all the extracts failed to inhibit the growth of most of the test fungi, significant antifungal activity against Cladosporium herbarum was exhibited by most notably the fruit (MID: 100 microg), and the leaf (MID: 400 microg) extracts. None of the extracts were significantly cytotoxic, and lethal towards brine shrimps. The root, leaf, trunk and stem bark extracts (except for the fruits) showed strong antioxidant activity exceeding that of the standard antioxidant, alpha-tocopherol. Antitumour-promoting activity (>95% inhibition) was shown by the fruit, leaf, stem and trunk bark extracts.
A 2-yr study of Japanese encephalitis (JE) virus in Sepang District, Selangor, Malaysia, was carried out to identify the mosquito vectors and to determine their seasonal abundance, parity, and infection rates. In total, 81,889 mosquitoes belonging to 9 genera and > 50 species were identified from CDC trap collections augmented with dry ice during 1992 and 1993. Culex tritaeniorhynchus Giles and Culex gelidus Giles were the most abundant species, and both increased in numbers with increases in rainfall. Overall, 45 JE virus isolations were made from 7 species-Cx. tritaeniorhynchus (24), Cx. gelidus (12), Culex fuscocephala Theobald (2), Aedes butleri Theobald (4), Culex quinquefasciatus Say (1), Aedes lineatopennis Ludlow (1), and Aedes (Cancraedes) sp. (1). Based on elevated abundance and JE infection rates, Cx. tritaeniorhynchus appears to be the most important vector of JE virus in Sepang.
The traditional methods used in the diagnosis of dengue infection do not lend themselves to field application. As such, clinical specimens have to be sent to a central laboratory for processing which invariably leads to delay. This affects patient management and disease control. The development of the dengue IgM dot enzyme immunoassay has opened up the possibility of carrying out the test in peripheral health settings.
Herbal medication has been practised by the rural Malaysian Malays for a long time. However, the long-term side-effects have never been studied. In the present study, 48 species of Euphorbiaceae were screened for tumour-promoter activity by means of an in vitro assay using a human lymphoblastoid cell line harbouring the Epstein-Barr virus (EBV) genome. Twenty-seven per cent (13 out of 48) of the species tested were found to be positive, and in four species, namely Breynia coronata Hk.f, Codiaeum variegatum (L) Bl, Euphorbia atoto and Exocoecaria agallocha, EBV-inducing activity was observed when the plant extracts were tested at low concentrations of between 0.2 and 1.2 micrograms ml-1 in cell culture. This observation warrants attention from the regular users of these plants because regular use of plants with tumour-promoting activity could well be an aetiological factor for the promotion of tumours among rural Malaysian Malays.
A cytotoxic T lymphocyte (CTL) response to dengue virus-infected target cells is described. Effector cells were generated in an in vitro secondary culture and appeared to be T cells possessing both the Lyt 1.1 and Lyt 2.1 surface antigens. A stronger CTL response was noted with the H-2k haplotype compared to H-2d, and H-2 compatibility was required between CTL and target cells. CTL generated showed some cross-reactivity with target cells infected with Japanese encephalitis virus (JEV), another flavivirus, but not with target cells infected with an alphavirus, Sindbis. The significance and importance of these findings are discussed.
The ability of passage in HeLa cells to attenuate flaviviruses was investigated for three different strains of the mosquito-borne West Nile (WN) virus and two tick-borne viruses, louping-ill and Langat. One strain of WN virus, Sarawak, was attenuated 4000-fold for adult mice by intraperitoneal or intranasal challenge after six HeLa passages. The HeLa-passaged virus was also found to be antigenically different and temperature-sensitive in its growth characteristics compared with the parent. After six HeLa cell passages the Egypt 101 and Smithburn strains of WN virus lost their ability to infect monkey kidney cells and no longer killed adult mice, although inoculated animals became sick for several days. In contrast, two tick-borne flaviviruses remained as virulent for mice after six HeLa passages as the parent non-HeLa-passaged virus. Neither of the tick-borne viruses exhibited characteristics associated with temperature sensitivity. The results, therefore, indicate that the mosquito-borne, but not tick-borne, flaviviruses can be attenuated by very few passages in HeLa cells. This observation may provide a model system with which to analyse the molecular basis of attenuation and/or virulence of mosquito-borne flaviviruses.
Different fatty acids have distinct effects on the survival of breast cancer cells, which could be mediated by fatty acid binding proteins (FABPs), a family of lipid chaperones. Due to the diverse structures of the members of FABP family, each FABP demonstrates distinct binding affinities to different fatty acids. Of note, FABP7 is predominantly expressed in triple negative breast cancer (TNBC), the most aggressive subtype of breast cancer. Yet, the role of FABP7 in modulating the effects of fatty acids on TNBC survival was unclear. In contrast to the high expression of FABP7 in human TNBC tumours, FABP7 protein was undetectable in TNBC cell lines. Hence, a FABP7 overexpression model was used for this study, in which the transduced TNBC cell lines (MDA-MB-231 and Hs578T) were treated with various mono- and polyunsaturated fatty acids. Oleic acid (OA), docosahexaenoic acid (DHA) and arachidonic acid (AA) inhibited TNBC cell growth at high concentrations, with no differences resulted from FABP7 overexpression. Interestingly, overexpression of FABP7 augmented linoleic acid-induced cell death in MDA-MB-231 cells. The increased cell death may be explained by a decrease in 13-HODE, a pro-tumorigenic oxidation product of linoleic acid. The phenotype was, however, attenuated with a rescue treatment using 25 nM 13-HODE. The decrease in 13-HODE was potentially due to fatty acid partitioning modulated by FABP7, as demonstrated by a 3-fold increase in fatty acid oxidation. Our findings suggest that linoleic acid could be a potential therapeutic strategy for FABP7-overexpressing TNBC patients.
Introduction: Bladder cancer is associated with high risk of tumour recurrence and therapeutic resistance. Cancer stem cells (CSC) within a particular tumour are postulated to drive tumorigenesis and influence tumour behaviour. Recent studies have shown that Newcastle disease virus (NDV) is able to selectively kill and exert a strong oncolytic effect against various cancer types. However little is known about the oncolytic effect of NDV against CSC. In this study, the oncolytic effect of NDV against putative bladder CSC was examined. Methods: Putative bladder CSC was selectively grown in the form of 3D-spheroids from six different bladder cancer cell lines. The spheroid cells were characterised for their stemness properties to ensure that these cells truly represent CSC. This was conducted via the analysis of CSC associated genes and cell surface markers expression. Subsequently, the oncolytic effect of the wild-type NDV-AF2240 strain against the bladder cancer spheroids was investigated. Results: All the spheroids expressed significantly high levels of CSC-associated genes. Flow-cytometry analysis revealed that the expression pattern of the CSC-associated surface markers was different in the spheroid cells; suggesting heterogeneity in the expression signatures of these cells. The infection of spheroids with NDV showed that the NDV was able to target bladder cancer spheroids but there was a spectrum of response across the different spheroids. Intriguingly, NDV was able to persistently infect bladder cancer spheroids that were not sensitive towards NDV infection as the presence of NDV viral genes were detected in the spheroid cells. The NDV persistently infected bladder cancer spheroids were resistant to superinfection and developed an antiviral state by expressing low levels of interferon-beta (IFN-b). NDV persistency of infection affects the process of epithelial to mesenchymal transition (EMT) of cancer cells as the spheroid forming ability of an established NDV persistently infected bladder cancer cell line, EJ28-PI was shown to be impaired. The EJ28-PI cells expressed significantly high levels of the EN2 gene. Knockdown of the EN2 expression reduced the viability of EJ28-PI cells; suggesting a role for EN2 in mediating NDV persistency of infection in cancer cells. Conclusion: Bladder CSC gene expression signatures influence the efficacy of NDV-mediated oncolysis. Our current work is focused on identifying genes and signalling pathways that influence NDV-mediated oncolysis us-ing whole-transcriptomic sequencing. The findings of this study can potentially be used to enhance the efficacy of NDV-mediated oncolysis and accelerate the translation of NDV as an oncotherapeutic agent in the clinic.
The Sumatran rhinoceros (Dicerorhinus sumatrensis), once widespread across Southeast Asia, now consists of as few as 30 individuals within Sumatra and Borneo. To aid in conservation planning, we sequenced 218 bp of control region mitochondrial (mt) DNA, identifying 17 distinct mitochondrial haplotypes across modern (N = 13) and museum (N = 26) samples. Museum specimens from Laos and Myanmar had divergent mtDNA, consistent with the placement of western mainland rhinos into the distinct subspecies D. s. lasiotis (presumed extinct). Haplotypes from Bornean rhinos were highly diverse, but dissimilar from those of other regions, supporting the distinctiveness of the subspecies D. s. harrissoni. Rhinos from Sumatra and Peninsular Malaysia shared mtDNA haplotypes, consistent with their traditional placement into a single subspecies D. s sumatrensis. Modern samples of D. s. sumatrensis were genotyped at 18 microsatellite loci. Rhinos within Sumatra formed 2 sub-populations, likely separated by the Barisan Mountains, though with only modest genetic differentiation between them. There are so few remaining Sumatran rhinoceros that separate management strategies for subspecies or subpopulations may not be viable, while each surviving rhino pedigree is likely to retain alleles found in no other individuals. Given the low population size and low reproductive potential of Sumatran rhinos, rapid genetic erosion is inevitable, though an under-appreciated concern is the potential for fixation of harmful genetic variants. Both concerns underscore 2 overriding priorities for the species: 1) translocation of wild rhinos to ex situ facilities, and 2) collection and storage of gametes and cell lines from every surviving captive and wild individual.
Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.