Imaging growth dynamics of tumour spheroids using optical coherence tomography

Optical coherence tomography (OCT) was used to monitor the dynamics of tumour spheroid formation by the hanging drop method. In contrast to microscopy, the estimates obtained using OCT for the volume of the spheroid, were consistent with the measured changes in cell number as a function of time. The OCT images also revealed heterogeneous structures in the spheroids of ∼200 μm diameter. These corresponded to the necrotic regions identified by fluorescence of propidium iodide stained cells.     

Plantlet production from the male inflorescence tips of <Emphasis Type="Italic">Musa acuminata</Emphasis> cultivars from South India

Inflorescence apices are suitable explants for the rapid in vitro propagation of Musa spp. However, the diploid and triploid banana cultivars showed different in vitro responses with respect to the hormone combinations in Murashige and Skoog medium. The diploid cultivar (Sannachenkadali, AA) induced a maximum number of multiple shoots in 8.9 μM 6-benzyl adenine (BA) whereas the triploid cultivar (Red banana, AAA) exhibited maximum multiplication in 22.2 μM 6-benzyl adenine. MS medium supplemented with 11.4 μM indole acetic acid and 17.8 μM BA was also suitable for shoot proliferation in triploid cultivar but not in the diploid cultivar. The regenerated shoots were rooted in Murashige and Skoog basal medium within 10–15 days. The rooted plantlets were transferred to vermiculite and maintained at a temperature of 25 ± 2°C for 10 days and then at room temperature (30–32°C) for 2 weeks before transferring to potted soil compost mixture. The plantlets showed 100% survival.     

DNA protective properties of vanillin against gamma-radiation under different conditions: possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of gamma-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to gamma-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of gamma-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, 'tail moment' and 'Olive tail moment'. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to gamma-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS*(+) radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against gamma-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.     

Optimizing cardiac material parameters with a genetic algorithm

Determining the unknown material parameters of intact ventricular myocardium can be challenging due to highly nonlinear material behavior. Previous studies combining a gradient-search optimization procedure with finite element analysis (FEA) were limited to two-dimensional (2D) models or simplified three-dimensional (3D) geometries. Here we present a novel scheme to estimate unknown material parameters for ventricular myocardium by combining a genetic algorithm (GA) with nonlinear finite element analysis. This approach systematically explores the domain of the material parameters. The objective function to minimize was the error between simulated strain data and finite element model strains. The proposed scheme was validated for a 2D problem using a realistic material law for ventricular myocardium. Optimized material parameters were generally within 0.5% of the true values. To demonstrate the robustness of the new scheme, unknown material parameters were also determined for a realistic 3D heart model with an exponential hyperelastic material law. When using strains from two material points, the algorithm converged to parameters within 5% of the true values. We conclude that the proposed scheme is robust when estimating myocardial material parameters in 2D and 3D models.     

Marek's disease virus type 2 (MDV-2)-encoded microRNAs show no sequence conservation with those encoded by MDV-1

MicroRNAs (miRNAs) are increasingly being recognized as major regulators of gene expression in many organisms, including viruses. Among viruses, members of the family Herpesviridae account for the majority of the currently known virus-encoded miRNAs. The highly oncogenic Marek's disease virus type 1 (MDV-1), an avian herpesvirus, has recently been shown to encode eight miRNAs clustered in the MEQ and LAT regions of the viral genome. The genus Mardivirus, to which MDV-1 belongs, also includes the nononcogenic but antigenically related MDV-2. As MDV-1 and MDV-2 are evolutionarily very close, we sought to determine if MDV-2 also encodes miRNAs. For this, we cloned, sequenced, and analyzed a library of small RNAs from the lymphoblastoid cell line MSB-1, previously shown to be coinfected with both MDV-1 and MDV-2. Among the 5,099 small RNA sequences determined from the library, we identified 17 novel MDV-2-specific miRNAs. Out of these, 16 were clustered in a 4.2-kb long repeat region that encodes R-LORF2 to R-LORF5. The single miRNA outside the cluster was located in the short repeat region, within the C-terminal region of the ICP4 homolog. The expression of these miRNAs in MSB-1 cells and infected chicken embryo fibroblasts was further confirmed by Northern blotting analysis. The identification of miRNA clusters within the repeat regions of MDV-2 demonstrates conservation of the relative genomic positions of miRNA clusters in MDV-1 and MDV-2, despite the lack of sequence homology among the miRNAs of the two viruses. The identification of these novel miRNAs adds to the growing list of virus-encoded miRNAs.     

Effect of seed soaking with sulphydryl compounds on the photochemical efficiency and antioxidant defence system during the growth of pearl millet under water limiting environment

Pearl millet (Pennisetum glaucum L. cv. HHB-67) seeds were pre-soaked in sulphydryl compounds (dithiothreitol, thioglycollic acid, thiourea, and cysteine). In plants at 59 and 67 d after sowing (DAS), activities of photosystem (PS) 2 (ferricyanide site) and PS1, both chloroplastic and total superoxide dismutase, glutathione reductase, and glutathione-S-transferase increased after all sulphydryl pre-treatments at both stages of plant development. Also dry matter of plant parts sampled at 55 DAS was higher after thiol-treatments in comparison with control.     

Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.     

Protective effect of n-3 polyunsaturated fatty acids concentrate on isoproterenol-induced myocardial infarction in rats

The protective effect of PUFA concentrate prepared from fish oil on isoproterenol-induced myocardial infarction in male albino rats was investigated with respect to changes in the levels of diagnostic marker enzymes, cholesterol, triglycerides, free fatty acids, phospholipids, reduced glutathione (GSH) and lipid peroxides (LPO). Administration of PUFA concentrate significantly prevented the isoproterenol-induced elevation in the levels of plasma diagnostic marker enzymes (ALT [93.5%], AST [95.6%], LDH [94.7%] and CPK [96.1%]). PUFA concentrate feeding exerted a significant antilipidemic effect against isoproterenol-induced myocardial infarction by reducing the levels of lipid components in plasma (cholesterol [71.5%], triglycerides [79.7%] and free fatty acids [70.7%] and heart tissue (cholesterol [81.4%], triglycerides [76.3%] and free fatty acids [78.6%]). A tendency to prevent the isoproterenol-induced phospholipids depletion (74.4%) in the myocardium of experimental rats was also observed. The level of lipid peroxidation was also found to be significantly lower in PUFA treated animals (2.72+/-0.15nmol/ml in plasma; 1.18+/-0.08nmol/mg protein in heart tissue) as compared to that of isoproterenol-injected groups (5.77+/-0.43nmol/ml in plasma; 2.14+/-0.15nmol/mg protein in heart tissue) of rats. Also the level of reduced GSH significantly higher in the heart tissue of PUFA administered experimental rats (5.65+/-0.98 microg/g) as compared to myocardial infarction induced control rats (2.39+/-0.18 microg/g). The results of the present study indicate that the overall cardioprotective effect of PUFA concentrate is probably related to its ability to inhibit lipid accumulation by its hypolipidaemic property.