Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs.
Hard structures of helminths have often been used for taxonomic identification but are usually not clearly defined when treated with conventional methods such as ammonium picrate-glycerin for monogeneans and glycerin for nematodes. The present study reports a rapid and simple technique to better resolve the hard parts of selected monogeneans and nematodes using 5-10% alkaline sodium dodecyl sulphate (SDS). In comparison with established methods, SDS-treated worms become more transparent. In monogeneans treated with SDS, clear details of the hooks, hook filaments, anchors, bars and the sclerotized copulatory organs could be observed. In SDS-treated nematodes, spicules and ornamentations of the buccal capsules could be clearly seen.
Image processing and analysis is an effective tool for monitoring and fault diagnosis of activated sludge (AS) wastewater treatment plants. The AS image comprise of flocs (microbial aggregates) and filamentous bacteria. In this paper, nine different approaches are proposed for image segmentation of phase-contrast microscopic (PCM) images of AS samples. The proposed strategies are assessed for their effectiveness from the perspective of microscopic artifacts associated with PCM. The first approach uses an algorithm that is based on the idea that different color space representation of images other than red-green-blue may have better contrast. The second uses an edge detection approach. The third strategy, employs a clustering algorithm for the segmentation and the fourth applies local adaptive thresholding. The fifth technique is based on texture-based segmentation and the sixth uses watershed algorithm. The seventh adopts a split-and-merge approach. The eighth employs Kittler's thresholding. Finally, the ninth uses a top-hat and bottom-hat filtering-based technique. The approaches are assessed, and analyzed critically with reference to the artifacts of PCM. Gold approximations of ground truth images are prepared to assess the segmentations. Overall, the edge detection-based approach exhibits the best results in terms of accuracy, and the texture-based algorithm in terms of false negative ratio. The respective scenarios are explained for suitability of edge detection and texture-based algorithms.
Aim: Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. Methods: In this investigation, the proliferation of brain tumor cell line, glioblastoma multiform (DBTRG.05MG) induced by NDV strain AF2240 was evaluated in-vitro, by using MTT proliferation assay. Furthermore, Cytological observations were studied using fluorescence microscopy and transmission electron microscopy, DNA laddering in agarose gel electrophoresis assay used to detect the mode of cell death and analysis of the cellular DNA content by flowcytometery. Results: MTT proliferation assay, Cytological observations using fluorescence microscopy and transmission electron microscopy show the anti-proliferation effect and apoptogenic features of NDV on DBTRG.05MG. Furthermore, analysis of the cellular DNA content showed that there was a loss of treated cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Conclusion: It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer.
Numerous specimens of Ancyrocephaloides triacanthi Yamaguti, 1938 and A. chauhani Bychowsky & Nagibina, 1975 were collected from two triacanthid fishes, Triacanthus biaculeatus and Tripodichthys blochii, off Peninsular Malaysia. The two monogenean species are redescribed and considered to be the only valid species of Ancyrocephaloides Yamaguti, 1938. Examinations of these worms revealed new features, e.g. the presence of exudates (both net-like and bundle-like) and superficial grooves in the anchors in both species, which necessitated re-descriptions of the two species and amendments to the generic diagnosis. Both species have relatively small anchors with two lateral superficial grooves along the shaft and point, peduncular glands and four large, pyriform secretory reservoirs in the peduncular-haptoral region, each with a single tubular extension to an associated anchor, and net-like structures (exudate) attached to the anchors. The net-like structures are one of the external manifestations of the secretion produced in the peduncular glands and stored in the pyriform secretory reservoirs. When released within the gill-tissue of the host, the exudate is in the form of bundles which extend within the gill-filament. The small anchors convey secretions from the secretory reservoirs via lateral superficial grooves into the gills as the anchors pierce the host tissue for attachment. The secretion coagulates as left and right thread-like bundles of exudate within the gill tissues and is only apparent as nets when it is released into the surrounding water. The recurved point of the anchor and position of the point of exudation allow the nets to remain attached to the anchor point, even after the detachment of the anchors from the gill tissue. This exudate possibly acts somewhat like a 'belay device' or 'safety belt', preventing the parasite from being washed away by the respiratory current during the onset of its leech-like locomotion, as well as assist the relatively small anchors in attachment.
Myliocotyle borneoensis sp. n. and M. multicrista sp. n. (Monocotylidae: Heterocotylinae) are described from the gills of the mottled eagle ray, Aetomylaeus maculatus (Gray), and the banded eagle ray A. nichofii (Bloch et Schneider) (Myliobatidae), respectively, collected from the northern coast of Malaysian Borneo. These are the first monogeneans to be described on elasmobranchs from Borneo. The formerly monotypic Myliocotyle (for M. pteromylaei) was distinguished from other monocotylids by the distribution and morphology of the eight sclerotised dorsal haptoral accessory structures and the morphology of the male copulatory organ. However, we have determined that M. pteromylaei has ten structures on the dorsal surface of the haptor. Myliocotyle borneoensis is distinguished from M. pteromylaei by the morphology of the male copulatory organ and its accessory piece. Myliocotyle multicrista has 12 sclerotised dorsal haptoral accessory structures and a male copulatory organ with two accessory pieces. Additional sclerotised ridges across the ventral surfaces of each loculus (except the posterior-most pair) are also present in M. multicrista. The diagnosis for Myliocotyle is revised given our discovery of additional dorsal haptoral accessory structures in the type species and to accommodate other new characters of the two new species. Anterior secretions of Myliocotyle are discussed.
The aim of this study was to determine the effect of butylated hydroxytoluene (BHT), a lipid-soluble anti-oxidant added in different concentrations to the Tris egg yolk extenders on semen cytological parameters pre freezing and post thawing (motility, morphology, viability, acrosome integrity and membrane integrity) of Boer goat spermatozoa. A total of 40 ejaculates from four Boer goat bucks were collected using an artificial vagina. Ten replicates of the ejaculates were diluted with a Tris egg yolk based extender which contained various concentrations (0.5mM, 1.0mM, 2.0mM and 3.0mM) of butylated hydroxytoluene while one sample was processed without supplementation of antioxidant and served as control. The diluted semen was cooled at 4°C and loaded into the straw and then stored in liquid nitrogen. It was evident that supplementation of BHT produces positive effect in terms of motility, membrane integrity and acrosome integrity in comparison with the control group in cooled and frozen Boer goat semen. Results showed significant differences in motility, membrane integrity, acrosome integrity and viability of cooled and frozen Boer goat spermatozoa at different concentrations. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group (P<0.05) while there was no significant differences (P>0.05) in morphology trait between all group in cooled semen. However, improvement (P<0.05) was observed only in terms of the membrane integrity and acrosome integrity compared to the control and other treated groups in frozen semen. In conclusion, BHT can be used in cryopreservation of Boer goat semen in order to reduce the oxidative stress on spermatozoa.
The current study focused on the microscopic studies of a native Bacillus thuringiensis strain isolated from Malaysia, Bt-S84-13a, that produced an unusual crystal type. Primary detection of parasporal inclusions using a phase contrast microscope presented one to two small crystal proteins in the sporulating cells of Bt-S84-13a. Compound light microscopic examination of autolysed Bt-S84-13a cells stained with 0.133% Coomassie Brilliant Blue showed two types of crystal morphology: small crystals independent of spores and spore-associated crystals. Surface structure analysis with a scanning electron microscope revealed spherical-like, coarse and wrinkled-looking crystal in Bt-S84-13a. A close-up observation of the crystal morphology using a transmission electron microscope also demonstrated two parasporal inclusions in Bt-S84-13a. One inclusion was deposited against the forespore and was in a shape of incomplete rectangular. Another smaller inclusion was developed within the exosporium and was rectangular in shape. However, the latter inclusion was found lack in another bacterial cell which was still in the early stages of sporulation. This unique crystal morphology may imply some biological potential in Bt-S84-13a.
Selections of collagen available commercially were tested for their biocompatibility as scaffold to promote cell growth in vitro via simple collagen fast test and cultivation of mammalian cells on the selected type of collagen. It was found that collagen type C9791 promotes the highest degree of aggregation as well as cells growth. This preliminary study also indicated potential use of collagen as scaffold in engineered tissue.
The value of urinary erythrocyte morphology in diagnosing glomerular and nonglomerular haematuria was studied using phase contrast microscopy in 105 patients with significant haematuria. Fifty-eight (93.6%) out of 62 patients with glomerulonephritis had dysmorphic erythrocytes and 40 (93.1%) out of the 43 patients with nonglomerular disease had isomorphic erythrocytes in the urine. A mixed picture of glomerular and nonglomerular haematuria was seen in 5 patients. The sensitivity was 93.6%, the specificity was 97.7% and the positive predictive value was 98.3% for glomerulonephritis in patients with dysmorphic haematuria. The positive predictive value for a nonglomerular source of bleeding was 96.7% with isomorphic haematuria. It is concluded that phase contrast microscopic examination of erythrocytes in urine is a simple, inexpensive and noninvasive technique that reliably distinguishes between glomerular and nonglomerular bleeding in patients.
This study was designed to evaluate the ability of natural coral implant to provide an environment for marrow cells to differentiate into osteoblasts and function suitable for mineralized tissue formation. DNA content, alkaline phosptatase (ALP) activity, calcium (Ca) content and mineralized nodules, were measured at day 3, day 7 and day 14, in rat bone marrow stromal cells cultured with coral discs glass discs, while cells alone and coral disc alone were cultured as control. DNA content, ALP activity, Ca content measurements showed no difference between coral, glass and cells groups at 3 day which were higher than control (coral disc alone), but there were higher measurement at day 7 and 14 in the cell cultured on coral than on glass discs, control cells and control coral discs. Mineralized nodules formation (both in area and number) was more predominant on the coral surface than in control groups. These results showed that natural coral implant provided excellent and favorable situation for marrow cell to differentiate to osteoblasts, lead to large amount of mineralized tissue formation on coral surface. This in vitro result could explain the rapid bone bonding of coral in vivo.
Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. In this investigation, the cytotolytic properties of NDV strain AF2240 were evaluated on brain tumor cell line, anaplastic astrocytoma (U-87MG), by using MTT assay. Cytological observations were studied using fluorescence microscopy and transmission electron microscopy to show the apoptogenic features of NDV on U-87MG. DNA laddering in agarose gel electrophoresis and terminal deoxyribonucleotide transferase-mediated dUTP-X nick end-labeling staining assay confirmed that the mode of cell death was by apoptosis. However, analysis of the cellular DNA content by flowcytometery showed that there was a loss of treated U-87MG cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Early apoptosis was observed 6 h post-inoculation by annexin-V flow-cytometry method. It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer.
We investigate induction of apoptosis by xanthohumol on Ca Ski cervical cancer cell line. Xanthohumol is a prenylated chalcone naturally found in hop plants, previously reported to be an effective anticancer agent in various cancer cell lines. The present study showed that xanthohumol was effective to inhibit proliferation of Ca Ski cells based on IC50 values using sulforhodamine B (SRB) assay. Furthermore, cellular and nuclear morphological changes were observed in the cells using phase contrast microscopy and Hoechst/PI fluorescent staining. In addition, 48-hour long treatment with xanthohumol triggered externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells. Additionally, xanthohumol mediated S phase arrest in cell cycle analysis and increased activities of caspase-3, caspase-8, and caspase-9. On the other hand, Western blot analysis showed that the expression levels of cleaved PARP, p53, and AIF increased, while Bcl-2 and XIAP decreased in a dose-dependent manner. Taken together, these findings indicate that xanthohumol-induced cell death might involve intrinsic and extrinsic apoptotic pathways, as well as downregulation of XIAP, upregulation of p53 proteins, and S phase cell cycle arrest in Ca Ski cervical cancer cells. This work suggests that xanthohumol is a potent chemotherapeutic candidate for cervical cancer.
Species of Phyllanthus have traditionally been used for hundreds of years for treating many ailments including diabetes, anemia, bronchitis and hepatitis. The present study aims to investigate the cytotoxic and apoptotic effects of methanol (PWM), hexane (PWH) and ethyl acetate (PWE) extracts from the leaves of the endemic plant Phyllanthus watsonii Airy Shaw (Phyllanthaceae) on MCF-7 human breast cancer cells. We observed that the PWM, PWH and PWE extracts were cytotoxic and selectively inhibited the growth and proliferation of MCF-7 cells compared to untreated control in a dose dependent manner with an IC(50) of 12.7 ± 4.65, 7.9 ± 0.60 and 7.7 ± 0.29 μg/ml, respectively. However, the extracts were not toxic at these concentrations to normal human lung fibroblast MRC-5 cells. Cell death induced by PWM, PWH and PWE extracts were mainly due to apoptosis which was characterized by apoptotic morphological changes and a nuclear DNA fragmentation. Caspase-3 activation following P. watsonii extracts treatment was also evident for apoptotic cell death which was preceded by an S phase cell cycle perturbation. The results suggested that the cytotoxic activity of P. watsonii extracts was related to an early event of cell cycle perturbation and a later event of apoptosis. Hence, P. watsonii displays potential to be further exploited in the discovery and development of new anticancer agents.