New detection system for toxic agents based on continuous spectroscopic monitoring of living cells

In this study, we show the feasibility of a new type of cell based biosensor which uses spectroscopic in situ real time detection of biochemical changes in living cells exposed to toxic chemical agents. We used a high power 785 nm laser to measure the time dependent changes in the Raman spectrum of individual living human lung cells (A549 cell line) treated with a toxic agent (Triton X-100, 250 microM solution). Individual cells were monitored by Raman spectroscopy over a total time span of 420 min, with 30 min sampling intervals. During this period of time, the A549 cells were maintained in a purpose designed temperature controlled cell chamber, which allowed the cells to be maintained in physiological conditions. The time dependent changes in the Raman spectra were correlated with the sequences of events that occur during cell death. The molecular mechanisms involved in cell death are indicated by the decrease in the magnitude of Raman peaks corresponding to proteins (1322, 1342 and 1005 cm(-1)) and DNA (decrease by 80-90% in the 786 cm(-1) phosphodiester bonds C'5-O-P-O-C'3). To support these conclusions, viability tests and Western blotting analysis of PARP protein were carried out. This technique could overcome the limitations of other detection systems available, since the specific time dependent biochemical changes in the living cells can be used for the identification and quantification of a large range of toxic agents. This technique could also be used with cellular microarrays for high throughput in vitro toxicological testing of pharmaceuticals and in situ monitoring of the growth of engineered tissues.     

The Effects of Hydrostatic Pressure on Living Aquatic Organisms V. Eurybiotic Environmental Capacity as a Factor in High Pressure Tolerance

Study on the total spectrum of organisms (72 species) subjected to hydrostatic pressure as of this date allows one to established categories of pressure tolerance (resistance): Extremely high – eurybiotic forms (1000–1200 atm), High – marine littoral, planktonic, freshwater (600–1000 atm), Moderate – marine littoral, planktonic, freshwater (400–600 atm), Low – planktonic, freshwater (100–300 atm), Extremely low – planktonic, freshwater (0–100 atm). The average pressure tolerance of marine littoral species is higher than that of planktonic species but not significantly different from freshwater species. Eurybiotic species which are not marine seem to show the highest pressure tolerance.     

Advancing Optical Imaging for Breast Margin Assessment: An Analysis of Excisional Time,Cautery, and Patent Blue Dye on Underlying Sources of Contrast

Breast conserving surgery (BCS) is a recommended treatment for breast cancer patients where the goal is to remove the tumor and a surrounding rim of normal tissue. Unfortunately, a high percentage of patients return for additional surgeries to remove all of the cancer. Post-operative pathology is the gold standard for evaluating BCS margins but is limited due to the amount of tissue that can be sampled. Frozen section analysis and touch-preparation cytology have been proposed to address the surgical needs but also have sampling limitations. These issues represent an unmet clinical need for guidance in resecting malignant tissue intra-operatively and for pathological sampling. We have developed a quantitative spectral imaging device to examine margins intra-operatively. The context in which this technology is applied (intra-operative or post-operative setting) is influenced by time after excision and surgical factors including cautery and the presence of patent blue dye (specifically Lymphazurin™, used for sentinel lymph node mapping). Optical endpoints of hemoglobin ([THb]), fat ([β-carotene]), and fibroglandular content via light scattering (<µ_s’>) measurements were quantified from diffuse reflectance spectra of lumpectomy and mastectomy specimens using a Monte Carlo model. A linear longitudinal mixed-effects model was used to fit the optical endpoints for the cautery and kinetics studies. Monte Carlo simulations and tissue mimicking phantoms were used for the patent blue dye experiments. [THb], [β-carotene], and <µ_s’> were affected by <3.3% error with <80 µM of patent blue dye. The percent change in [β-carotene], <µ_s’>, and [β-carotene]/<µ_s’> was <14% in 30 minutes, while percent change in [THb] was >40%. [β-carotene] and [β-carotene]/<µ_s’> were the only parameters not affected by cautery. This work demonstrates the importance of understanding the post-excision kinetics of ex-vivo tissue and the presence of cautery and patent blue dye for breast tumor margin assessment, to accurately interpret data and exploit underling sources of contrast.     

Sorption of plutonium, americium and fission products from reprocessing effluents using Rhizopus arrhizus

Rhizopus arrhizus biomass removed more than 95% of 239Pu, 241Am, 95Zr, 144Ce and 152+154Eu from different waste streams generated in Purex as well as Truex processes after suitable adjustment of pH. © Rapid Science Ltd. 1998     

Design of grasping methodologies for rotational assembly components

This article describes a design methodology for grasping assembly components. Three heuristics are developed. The first determines feasible grasping configurations based on component geometric information. The second heuristic determines feasible grasping configurations by including gripper functional attributes. The third heuristic generates the final set of grasping configurations by including the area available for grasping a component. An interactive program written in Fortran 77 is developed to capture the user inputs, and a sample application is described. The methodology does not assume an initial feeding state of the component to the robot. The grasping configurations generated (if more than one) provide the designer with alternate feasible feeding methods.     

Dietary gangliosides increase the content and molecular percentage of ether phospholipids containing 20:4n-6 and 22:6n-3 in weanling rat intestine

This study was conducted to determine whether dietary ganglioside (GG) increases the content of ether phospholipids (EPL) in intestinal mucosa. Weanling Sprague-Dawley rats were fed a semipurified diet consisting of 20% fat as a control diet. Two experimental diets were formulated by adding either 0.1% (w/w fat) GGs (GG diet) or 1.0% (w/w fat) sphingomyelin (SM diet) to the control diet. Fatty acid methyl esters from the alkenylacyl, alkylacyl and diacyl subclasses of phospholipids were measured to determine total and molecular percentage of EPL comprising the choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG) fraction. Animals fed the GG diet significantly increased total EPL content both in CPG (by 36%) and in EPG (by 66%), and the molecular percentage of EPL in CPG (by 76%) and in EPG (by 59%) compared to animals fed the control diet. Dietary GG-induced increase in EPL resulted in a higher level of polyunsaturated fatty acids (PUFA) specifically in 20:4n-6 and 22:6n-3 compared to control animals, leading to a decrease in the ratio of saturated fatty acids (SFA) to PUFA both in CPG and in EPG. Feeding animals the SM diet showed a higher level of EPL than control animals with a concomitant increase in 22:6n-3 in EPL. The present data demonstrate that dietary GG increases the content and composition of EPL containing PUFA in the weanling rat intestine.     

In vitro polymerization of Mycobacterium leprae FtsZ OR Mycobacterium tuberculosis FtsZ is revived or abolished, respectively, by reciprocal mutation of a single residue

A single residue that dramatically influences polymerization of principal cell division protein FtsZ of Mycobacterium leprae (MlFtsZ) and Mycobacterium tuberculosis (MtFtsZ) has been identified. Soluble, recombinant MlFtsZ did not show polymerization in vitro, in contrast to MtFtsZ, which polymerised. Mutation of the lone non-conserved residue T172 in the N-terminal domain of MlFtsZ to A172, as it exists in MtFtsZ, showed dramatic polymerization of MlFtsZ-T172A in vitro. Reciprocal mutation of A172 in MtFtsZ to T172, as it exists in MlFtsZ, abolished polymerization of MtFtsZ-A172T in vitro. While T172A mutation enhanced weak GTPase activity of MlFtsZ, reciprocal A172T mutation marginally reduced GTPase activity of MtFtsZ in vitro. These observations demonstrate that the residue at position 172 plays critical role in the polymerization of MlFtsZ and MtFtsZ. A possible evolutionary correlation between the presence of polymerization-adversive or polymerization-favouring residue at position 172 in FtsZ and generation time of the respective bacterium are discussed.     

Improvement of Biomass Partitioning, Flowering and Yield by Triadimefon in UV-B Stressed Vigna radiata (L.) Wilczek

Elevated UV-B radiation (12.2 kJ m^–2 d^–1) as against the ambient level of 10 kJ m^–2 d^–1 affected flowering, productivity and biomass partitioning of green gram [Vigna radiata (L.) Wilczek cv. KM-2]. UV-B stress delayed flowering initiation and achievement of 50 % flowering, reduced flower retention by 25 %, potential yield by 18 % and all yield attributes such as pod number (25 %), pod mass (41 %), seed number (32 %) and seed mass (45 %). Harvest index and shelling percentage were also reduced by 31 and 7 %, respectively. Application of triadimefon (20 mg dm^–3) to unstressed plants accelerated flowering and enhanced flower retention (21 %), potential yield (15 %) and yield attributes (7 to 44 %). The partitioning of biomass between plant parts also showed improvement over the control plants. In UV-B-stressed plants, triadimefon treatment compensated the inhibitions to varying extents.     

Analysis of degradation of bacterial cell division protein FtsZ by the ATP-dependent zinc-metalloprotease FtsH in vitro

The identity of protease(s), which would degrade bacterial cell division protein FtsZ in vivo, remains unknown. However, we had earlier demonstrated that Escherichia coli metalloprotease FtsH degrades E. coli cell division protein FtsZ in an ATP- and Zn(2+)-dependent manner in vitro. In this study, we examined FtsH protease-mediated degradation of FtsZ in vitro in detail using seven different deletion mutants of FtsZ as the substrates, which lack different extents of specific regions at the N- or C-terminus. FtsH protease assay in vitro on these mutants revealed that FtsH could degrade all the seven deletion mutants irrespective of the deletions or the extent of deletions at the N- or C-terminus. These observations indicated that neither the N-terminus nor the C-terminus was required for the degradation of FtsZ, like already known in the case of the FtsH substrate sigma(32) protein. The recombinant clones expressing full-length FtsZ protein and FtsZ deletion mutant proteins would be useful in investigating the possibility of FtsZ as a potential in vivo substrate for FtsH in ftsH-null cells carrying ftsH suppressor function and ectopically expressed FtsH protease.     

Phenotypic and phylogenic groups to evaluate the diversity of Citrobacter isolates from activated biomass of effluent treatment plants

The diversity of Citrobacter isolated from effluent treatment plants (ETPs) was studied using three different parameters. Thirty Citrobacter strains were isolated from different ETPs treating wastewaters generated at various industries. All the isolates were characterized based on biochemical tests, antibiotic assay/functional analysis, and phylogenetic analysis. Results demonstrated that the pattern of grouping varied based on the selected criteria for analysis. Species that clustered together by biochemical analysis were found to vary by functional and 16S rDNA analysis and vice versa. This suggests that multiple methods approach needs to be carried out to understand the microbial diversity. Bacteria in effluent treatment plants are exposed to diverse categories of pollutants. Salicylate is a key intermediate formed during biodegradation of several aromatic compounds, a scenario expected in ETPs. Hence, the Citrobacter isolates were screened for their capability to utilize salicylate. In future studies, these isolates can be incorporated in a bioremediation program.