Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

Striped catfish (Pangasianodon hypophthalmus) could be suitable for coastal aquaculture

Salinity intrusion in the coastal freshwater rivers due to climate change and construction of the dam in the upstream rivers are alarming in aquaculture. Hence, an experiment was conducted to know the effects of salinity on growth performance, hemato‐biochemical parameters and erythrocytes structure in a commercially cultivable catfish species, striped catfish (Pangasianodon hypophthalmus). Firstly, median lethal concentration (LC₅₀) of salinity for striped catfish was determined and then the fish were exposed to three salinity conditions (4, 8 and 12‰) and a control (0‰). Fish were sacrificed at day 7, 14, 28 and 56 after the start of salinity exposure. The 96 hr LC₅₀ value was found to be 14.87‰. Salinity levels from freshwater to 8‰ showed optimal conditions with high survival rate and good growth performances of fish in terms of weight gain and specific growth rate (SGR). Interestingly, the lowest food conversion ratio (FCR) was found in 4‰ group. The hemoglobin (Hb) level and number of red blood cells (RBCs) were found to be decreased significantly in 8 and 12‰ compared to 0 and 4‰ at the initial days of exposure, while number of white blood cells (WBCs) and glucose level showed opposite scenario. Frequencies of ENA (erythrocytic nuclear abnormalities) and ECA (erythrocytic cellular abnormalities) were significantly increased with increasing salinities in the initial days of exposure. Overall, findings of the present study revealed that striped catfish might be suitable fish species for culture in the brackish water containing salinity up to 10‰.     

Evolution of resistance to Aurora kinase B inhibitors in leukaemia cells

Aurora kinase inhibitors are new mitosis-targeting drugs currently in clinical trials for the treatment of haematological and solid malignancies. However, knowledge of the molecular factors that influence sensitivity and resistance remains limited. Herein, we developed and characterised an in vitro leukaemia model of resistance to the Aurora B inhibitor ZM447439. Human T-cell acute lymphoblastic leukaemia cells, CCRF-CEM, were selected for resistance in 4 μM ZM447439. CEM/AKB4 cells showed no cross-resistance to tubulin-targeted and DNA-damaging agents, but were hypersensitive to an Aurora kinase A inhibitor. Sequencing revealed a mutation in the Aurora B kinase domain corresponding to a G160E amino acid substitution. Molecular modelling of drug binding in Aurora B containing this mutation suggested that resistance is mediated by the glutamate substitution preventing formation of an active drug-binding motif. Progression of resistance in the more highly selected CEM/AKB8 and CEM/AKB16 cells, derived sequentially from CEM/AKB4 in 8 and 16 μM ZM447439 respectively, was mediated by additional defects. These defects were independent of Aurora B and multi-drug resistance pathways and are associated with reduced apoptosis mostly likely due to reduced inhibition of the catalytic activity of aurora kinase B in the presence of drug. Our findings are important in the context of the use of these new targeted agents in treatment regimes against leukaemia and suggest resistance to therapy may arise through multiple independent mechanisms.     

Effects of age,replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.     

Regional and species differences in endogenous prostaglandin biosynthesis by brain homogenates.

Endogenously formed prostaglandins (PGs) D2, E2 and F2 alpha were determined in homogenates of brain regions from rat, guinea-pig, rabbit and cat, using gas-chromatography-mass spectrometry. The main PGs formed in the brain regions of the rat were PGD2, in the guinea-pig PGD2 and PGF2 alpha, in the rabbit PGF2 alpha and in the cat PGE2. Brain regions from the same animal species showed the same pattern of PG formation. They varied, however, in the amount of total PGs formed, the limbic system and the cerebral cortex being highest and cerebellum lowest.     

Characterization of AmphiF-spondin reveals the modular evolution of chordate F-spondin genes

The F-spondin genes are a family of extracellular matrix molecules united by two conserved domains, FS1 and FS2, at the amino terminus plus a variable number of thrombospondin repeats at the carboxy terminus. Currently, characterized members include a single gene in Drosophila and multiple genes in vertebrates. The vertebrate genes are expressed in the midline of the developing embryo, primarily in the floor plate of the neural tube. To investigate the evolution of chordate F-spondin genes, I have used the basal position in chordate phylogeny of the acraniate amphioxus. A single F-spondin-related gene, named AmphiF-spondin, was isolated from amphioxus. Based on molecular phylogenetics, AmphiF-spondin is closely related to a particular subgroup of vertebrate F-spondin genes that encode six thrombospondin repeats. However, unlike these genes, expression of AmphiF-spondin is not confined to the midline but is found through most of the central nervous system. Additionally, AmphiF-spondin has lost three thrombospondin repeats and gained two fibronectin type III repeats, one of which has strong identity to a fibronectin type III repeat from Deleted in Colorectal Cancer (DCC). Taken together, these results suggest a complex evolutionary history for chordate F-spondin genes that includes (1) domain loss, (2) domain gain by tandem duplication and divergence of existing domains, and (3) gain of heterologous domains by exon shuffling.