Influence of nutrient supply and elicitors on glucosinolate production in <Emphasis Type="Italic">E. sativa</Emphasis> hairy root cultures

Glucosinolates (GS) are secondary plant metabolites comprising different subgroups with opposing effects on human health. Hairy root cultures (HRC) are potent biotechnological tools allowing the biosynthesis of special substances under defined conditions. HRC of Eruca sativa, a brassicaceaous plant, were used to test different strategies to enhance GS levels and to alter the profile. Additional sulphur supply in the nutrient medium increased especially aliphatic GS by 2.7-fold, but also enhanced indole GS by 1.8-fold. Ethephon as well as jasmonic acid as chemical elicitors enhanced only indole GS levels, whereby especially 4-methoxyindol-3-ylmethyl or 1-methoxyindol-3-ylmethyl GS accumulated. Jasmonic acid was used in combination with pulsed electric field treatment as physical elicitor. Already within 24 h, GS levels doubled in treated HRC compared to the control. For estimation of production potency, the GS levels of HRC were compared to contents of aerial and root parts of E. sativa sprouts. HRC showed a distinct GS profile compared to the parent plant with a higher content of indole GS when compared to sprout roots, but overall lower total GS levels. Furthermore, HRC released GS into the culture medium, which could be enhanced by jasmonic acid and pulsed electric field treatment. This could comprise an efficient strategy for a continuous GS production and mining without solvent extraction.     

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.     

Pharmacologic modulation of RNA splicing enhances anti-tumor immunity

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Bayesian-based selection of metabolic objective functions

MOTIVATION: A critical component of in silico analysis of underdetermined metabolic systems is the identification of the appropriate objective function. A common assumption is that the objective of the cell is to maximize growth. This objective function has been shown to be consistent in a few limited experimental cases, but may not be universally appropriate. Here a method is presented to quantitatively determine the most probable objective function. RESULTS: The genome-scale metabolism of Escherichia coli growing on succinate was used as a case-study for analysis. Five different objective functions, including maximization of growth rate, were chosen based on biological plausibility. A combination of flux balance analysis and linear programming was used to simulate cellular metabolism, which was then compared to independent experimental data using a Bayesian objective function discrimination technique. After comparing rates of oxygen uptake and acetate production, minimization of the production rate of redox potential was determined to be the most probable objective function. Given the appropriate reaction network and experimental data, the discrimination technique can be applied to any bacterium to test a variety of different possible objective functions. SUPPLEMENTARY INFORMATION: Additional files, code and a program for carrying out model discrimination are available at http://www.engr.uconn.edu/~srivasta/modisc.html.     

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.Download video file.^{(95M, mov)}     

Human pluripotent stem cells produce natural killer cells that mediate anti-HIV-1 activity by utilizing diverse cellular mechanisms

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication, their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly, both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis, antibody-dependent cellular cytotoxicity, and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS.     

How the molecular structure determines the flow of excitation energy in plant light-harvesting complex II

Excitation energy transfer in the light-harvesting complex II of higher plants is modeled using excitonic couplings and local transition energies determined from structure-based calculations recently (Müh et al., 2010). A theory is introduced that implicitly takes into account protein induced dynamic localization effects of the exciton wavefunction between weakly coupled optical and vibronic transitions of different pigments. Linear and non-linear optical spectra are calculated and compared with experimental data reaching qualitative agreement. High-frequency intramolecular vibrational degrees of freedom are found important for ultrafast subpicosecond excitation energy transfer between chlorophyll (Chl) b and Chla, since they allow for fast dissipation of the excess energy. The slower ps component of this transfer is due to the monomeric excited state of Chlb 605. The majority of exciton relaxation in the Chla spectral region is characterized by slow ps exciton equilibration between the Chla domains within one layer and between the lumenal and stromal layers in the 10-20 ps time range. Subpicosecond exciton relaxation in the Chla region is only found within the terminal emitter domain (Chls a 610/611/612) and within the Chla 613/614 dimer. Deviations between measured and calculated exciton state life times are obtained for the intermediate spectral region between the main absorbance bands of Chla and Chlb that indicate that besides Chlb 608 another pigment should absorb there. Possible candidates, so far not identified by structure-based calculations, but by fitting of optical spectra and mutagenesis studies, are discussed. Additional mutagenesis studies are suggested to resolve this issue.     

Permafrost thaw causes large carbon loss in boreal peatlands while changes to peat quality are limited

Rapid, ongoing permafrost thaw of peatlands in the discontinuous permafrost zone is exposing a globally significant store of soil carbon (C) to microbial processes. Mineralization and release of this peat C to the atmosphere as greenhouse gases is a potentially important feedback to climate change. Here we investigated the effects of permafrost thaw on peat C at a peatland complex in western Canada. We collected 15 complete peat cores (between 2.7 and 4.5 m deep) along four chronosequences, from elevated permafrost peat plateaus to saturated thermokarst bogs that thawed up to 600 years ago. The peat cores were analysed for peat C storage and peat quality, as indicated by decomposition proxies (FTIR and C/N ratios) and potential decomposability using a 200-day aerobic laboratory incubation. Our results suggest net C loss following thaw, with average total peat C stocks decreasing by ~19.3 ± 7.2 kg C m⁻² over <600 years (~13% loss). Average post-thaw accumulation of new peat at the surface over the same period was ~13.1 ± 2.5 kg C m⁻². We estimate ~19% (±5.8%) of deep peat (>40 cm below surface) C is lost following thaw (average 26 ± 7.9 kg C m⁻² over <600 years). Our FTIR analysis shows peat below the thaw transition in thermokarst bogs is slightly more decomposed than peat of a similar type and age in permafrost plateaus, but we found no significant changes to the quality or lability of deeper peat across the chronosequences. Our incubation results also showed no increase in C mineralization of deep peat across the chronosequences. While these limited changes in peat quality in deeper peat following permafrost thaw highlight uncertainty in the exact mechanisms and processes for C loss, our analysis of peat C stocks shows large C losses following permafrost thaw in peatlands in western Canada.