Systems Analysis of the Response of Photosynthesis, Metabolism, and Growth to an Increase in Irradiance in the Photosynthetic Model Organism Chlamydomonas reinhardtii.

We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance.

Diurnal changes of polysome loading track sucrose content in the rosette of wild-type arabidopsis and the starchless pgm mutant.

Growth is driven by newly fixed carbon in the light, but at night it depends on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night, and low CO2 in Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) and in the starchless phosphoglucomutase (pgm) mutant. In Col-0, polysome loading was 60% to 70% in the light, 40% to 45% for much of the night, and less than 20% in an extended night, while in pgm, it fell to less than 25% early in the night. Quantification of ribosomal RNA species using quantitative reverse transcription-polymerase chain reaction revealed that polysome loading remained high for much of the night in the cytosol, was strongly light dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared with Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from (13)CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modeling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in the previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon.

Assessment of Accuracy of Implant Placement Using Surgical Stent Versus Conventional Technique.

Purpose: The aim of this study was to assess the accuracy of the implant placement using surgical stents versus conventional implant placement. Materials and Methods: Twenty patients who reported to Rajarajeswari Dental College and Hospital were selected and they were randomly divided into two groups. In both the groups, the selection of the implant size was based on the ridge mapping. In Group I, tooth and tissue-supported surgical stent was fabricated and was used to place the implant. In Group II conventional technique was used for implant placement. Results: In Group I the implant placement was parallel to the adjacent tooth with less variation in the angulation and the mesiodistal orientation of the implant was statistically significant compared to Group II. Conclusion: This technique of stent fabrication is easy, reliable, and cost-effective which also aids in the ideal placement of the implant in terms of orientation and angulation.

Evaluation of BBB permeable nucleolipid (NLDPU): A di-C15-ketalised palmitone appended uridine as neuro-tracer for SPECT.

Despite being in routine for onco-diagnostics for years, the applicability of nucleosidic molecular imaging probes is severely restricted in neurological applications due to their low permeability across blood-brain-barrier (BBB). For extending nucleoside tracers utility for neuro-onco early diagnostics, suitable modification which enhances their BBB permeation needs investigation. Among various modifications, lipidization of nucleosides has been reported to enhance cellular permeability. Extending the concept, the aim was to exemplify the possibility of lipidized nucleosides as potential brain tracer with capability to cross intact BBB and evaluate as metal based neuro-imaging SPECT agent. Uridine based non-lipidic (NSDAU) and di-C15-ketal appended lipidic (NLDPU) ligands were conjugated to chelator, DTPA (DTPA-NSDAU and DTPA-NLDPU) using multi-step chemistry. The ligands were evaluated in parallel for comparative physical and biological parameters. Additionally, effects of enhanced lipophilicity on UV-absorption, acid strength, fluorescence and non-specific protein binding were evaluated. Fluorescence quenching of BSA indicated appreciable interaction of DTPA-NLDPU with protein only above 10 mM without inducing conformational changes. In addition, DTPA-NLDPU was found to be haematocompatible and cytocompatible with low dose-dependent toxicity in HEK-cells. The chelator DTPA was used for 99mTc-complexation for SPECT imaging. Optimized 99mTc-radiolabeling parameters resulted in quantitative (≥97%) labeling with good stability parameters in in-vitro serum and cysteine challenge studies. We demonstrate that the nucleolipid radiotracer (99mTc-DTPA-NLDPU) was successfully able to permeate the BBB with brain uptake of 0.2% ID/g in normal mice as compared to 0.06% ID/g uptake of 99mTc-DTPA-NSDAU at 5 min. Blood kinetics indicate biphasic profile and t1/2(distribution) 46 min for 99mTc-DTPA-NLDPU. The preferential accumulation of 99mTc-DTPA-NLDPU in brain tumor intracranial xenograft indicate the targeting capability of the nucleoside. We conclude that as first-of-its-kind, this work presents the potential of the biocompatible nucleolipidic system for brain targeting and early diagnostics.

Electrical breakdown gas detector featuring carbon nanotube array electrodes.

We demonstrate here detection of dichloro-difluoro-methane and oxygen in mixtures with helium using a carbon nanotube electrical breakdown sensor device. The sensor is comprised of an aligned array of multiwalled carbon nanotubes deposited on a nickel based super-alloy (Inconel 600) as the anode; the counter electrode is a planar nickel sheet. By monitoring the electrical breakdown characteristics of oxygen and dichloro-difluoro-methane in a background of helium, we find that the detection limit for dichloro-difluoro-methane is approximately 0.1% and the corresponding limit for oxygen is approximately 1%. A phenomenologigal model is proposed to describe the trends observed in detection of the two mixtures. These results indicate that carbon nanotube based electrical breakdown sensors show potential as end detectors in gas-chromatography devices.

Improvising 5-HT7R homology model for design of high affinity ligands: model validation with docking, embrace minimization, MM-GBSA, and molecular dynamic simulations.

The subtype, 5-HT7R has been implicated in neurological disorders and presents itself as a promising target for antidepressant drugs. Design of targeted selective ligands, require a sound knowledge of 3D-receptor structure. In absence of receptor structure, structure-based design of targeted ligands relies on generation of 5-HT7R homology model. In this study, the impact of template choice, alignment, and model building methods on the homology model of 5-HT7R is addressed. The compactness and model quality due to the presence of cholesterol (lipidic receptor) have also been observed. The results suggest good stereochemical quality of the final model. Ramachandran Plot Analysis indicated more than 97.5% amino acid residues in the favorable region. The overall quality factor was 91.8% using ERRAT. The Z-score for backbone confirmation and packing quality were -1.248 and -1.427, respectively, using WHATCHECK. The RMS Z-score for side chain planarity was .711. Other validation results for the final model include binding site analysis in which Asp162, Val163, Phe343, Phe344, Arg350, Arg367, and Leu370 conserved residues were found in the active site, correlation coefficient of .82 in ligand-based screening and .85 in embrace minimization. Further, the model showed good correlation for agonist and antagonist in docking ([Formula: see text] ≈ .76, [Formula: see text] ≈ .82), embrace minimization ([Formula: see text] ≈ .73, [Formula: see text] ≈ .72), and MM-GBSA ([Formula: see text] ≈ .69, [Formula: see text] ≈ .75) studies. The model was subjected to Molecular Dynamics (MD) simulation of 20 ns both in ligand-free and ligand-bound receptor (agonist and antagonist) system in order to assess its stability.

Evidence for altered metabolic pathways during environmental stress: (1)H-NMR spectroscopy based metabolomics and clinical studies on subjects of sea-voyage and Antarctic-stay.

The Antarctic context is an analogue of space travel, with close similarity in ambience of extreme climate, isolation, constrained living spaces, disrupted sleep cycles, and environmental stress. The present study examined the impact of the harsh habitat of Antarctica on human physiology and its metabolic pathways, by analyzing human serum samples, using (1)H-NMR spectroscopy for identification of metabolites; and quantifying other physiological and clinical parameters for correlation between expression data and metabolite data. Sera from seven adult males (of median age 36years) who participated in this study, from the 28th Indian Expeditionary group to the Antarctica station Maitri, were collected in chronological sequence. These included: i) baseline control; ii) during ship journey; iii) at Antarctica, in the months of March, May, August and November; to enable study of temporal evolution of monitored physiological states. 29 metabolites in serum were identified from the 400MHz (1)H-NMR spectra. Out of these, 19 metabolites showed significant variations in levels, during the ship journey and the stay at Maitri, compared to the base-line levels. Further biochemical analysis also supported these results, indicating that the ship journey, and the long-term Antarctic exposure, affected kidney and liver functioning. Our metabolite data highlights for the first time the effect of environmental stress on the patho-physiology of the human system. Multivariate analysis tools were employed for this metabonomics study, using (1)H-NMR spectroscopy.

Design and synthesis of calcium responsive magnetic resonance imaging agent: Its relaxation and luminescence studies.

Calcium concentration modulation both inside and outside cell is of considerable interest for nervous system function in normal and pathological conditions. MRI has potential for very high spatial resolution at molecular/cellular level. Design, synthesis and evaluation of Gd-DO3A-AME-NPHE, a calcium responsive MRI contrast agent is presented. The probe is comprised of a Gd(3+)-DO3A core coupled to iminoacetate coordinating groups for calcium induced relaxivity switching. In the absence of Ca(2+) ions, inner sphere water binding to the Gd-DO3A-AME-NPHE is restricted with longitudinal relaxivity, r1 = 4.37 mM(-1) s(-1) at 4.7 T. However, addition of Ca(2+) triggers a marked enhancement in r1 = 6.99 mM(-1) s(-1) at 4.7 T (60% increase). The construct is highly selective for Ca(2+) over competitive metal ions at extracellular concentration. The r1 is modulated by changes in the hydration number (0.2 to 1.05), which was confirmed by luminescence emission lifetimes of the analogous Eu(3+) complex. T1 phantom images establish the capability of complex of visualizing changes in [Ca(2+)] by MRI.

Solid phase synthesis, radiolabeling and biological evaluation of a (99m) Tc-labeled α(V)ß(3) tripeptide (RGD) conjugated to DOTA as a tumor imaging agent.

Solid phase synthesis of peptides-radiometal chelator can facilitate the creation of radioactive peptide libraries to be utilized in high throughput in in vivo screening of targeted molecular imaging agents. An α(V)ß(3) tripeptide derivative DOTA-NH-Arg-Gly-Asp was synthesized by Fmoc solid phase peptide synthesis and analyzed by spectroscopic techniques. In order to radiolabel this RGD peptide with (99m)Tc-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was incorporated as a chelator. The DOTA-peptide conjugate binds to (99m)Tc with high efficiency at ambient temperature. The resulting conjugate is stable under physiological conditions for at least 24 h after radiocomplexation. The receptor binding studies of (99m)Tc-DOTA- α(V)ß(3)-tripeptide in established human tumor cell lines U-87MG and BMG revealed K(d) values in the nM and µM range respectively. U-87MG tumors in athymic mice were accumulated in the γ-images and major accumulation of the radiotracer was observed in kidneys followed by liver and lungs. High tumor uptake was shown in the U-87MG tumor bearing athymic mice; tumor to muscle ratios reached 8.13 ± 2.18 and 35.09 ± 4.78 at 1 and 4 h after post injection respectively.

A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.).

A robust and high throughput Agrobacterium genetic transformation procedure has been developed for perennial ryegrass (Lolium perenne L.). Embryogenic callus lines were selected and maintained as plants in vitro. Embryogenic calli derived from meristematic regions of the vegetative tillers were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying the plasmid pCAMBIA 1305.1 in the presence of acetosyringone for 3-4 days. The calli were grown under 94.8 and 151.6 microM hygromycin selection, respectively for two cycles of 2-weeks each, followed by transfer to regeneration medium with 47.4 microM hygromycin. Regenerated plants were rooted and successfully transferred to soil. The transgenic nature of the regenerated plants was confirmed by DNA gel blot analysis and gene expression demonstrated by GUS histochemical assay and/or reverse transcription PCR. After development of the transformation procedure, we used Agrobacterium strain EHA101 carrying a modified binary plasmid pMH bearing genes of interest. In the past 2 years, we have produced more than 1,000 plants with constructs encoding different genes of interest from perennial ryegrass.