Mapping sub-cellular protein aggregates and lipid inclusions using synchrotron ATR-FTIR microspectroscopy.

Visualising direct biochemical markers of cell physiology and disease pathology at the sub-cellular level is an ongoing challenge in the biological sciences. A suite of microscopies exists to either visualise sub-cellular architecture or to indirectly view biochemical markers (e.g. histochemistry), but further technique developments and innovations are required to increase the range of biochemical parameters that can be imaged directly, in situ, within cells and tissue. Here, we report our continued advancements in the application of synchrotron radiation attenuated total reflectance Fourier transform infrared (SR-ATR-FTIR) microspectroscopy to study sub-cellular biochemistry. Our recent applications demonstrate the much needed capability to map or image directly sub-cellular protein aggregates within degenerating neurons as well as lipid inclusions within bacterial cells. We also characterise the effect of spectral acquisition parameters on speed of data collection and the associated trade-offs between a realistic experimental time frame and spectral/image quality. Specifically, the study highlights that the choice of 8 cm-1 spectral resolutions provide a suitable trade-off between spectral quality and collection time, enabling identification of important spectroscopic markers, while increasing image acquisition by ∼30% (relative to 4 cm-1 spectral resolution). Further, this study explores coupling a focal plane array detector with SR-ATR-FTIR, revealing a modest time improvement in image acquisition time (factor of 2.8). Such information continues to lay the foundation for these spectroscopic methods to be readily available for, and adopted by, the biological science community to facilitate new interdisciplinary endeavours to unravel complex biochemical questions and expand emerging areas of study.

Complementary Approaches to Imaging Subcellular Lipid Architectures in Live Bacteria Using Phosphorescent Iridium Complexes and Raman Spectroscopy.

A family of three neutral iridium(III) tetrazolato complexes are investigated as bacterial imaging agents. The complexes offer a facile tuning of the emission colour from green (520 nm) to red (600 nm) in aqueous media, while keeping the excitation wavelength unchanged. The three complexes do not inhibit the bacterial growth of Bacillus Cereus, used as a model in this study, and exhibit extremely fast cellular uptake. After a minute incubation time, the nontoxic complexes show subcellular localisation in spherical structures identified as lipid vacuoles. Confocal Raman imaging has been exploited for the first time on live bacteria, to provide direct and label-free mapping of the lipid-enriched organelles within B. cereus, complementing the use of luminescent probes. Examination of the Raman spectra not only confirmed the presence of lipophilic inclusions in B. cereus but offered additional information about their chemical composition, suggesting that the lipid vacuoles may contain polyhydroxybutyrate (PHB).

Methylation of Ir(iii)-tetrazolato complexes: an effective route to modulate the emission outputs and to switch to antimicrobial properties.

Two neutral cyclometalated Ir(iii)-tetrazolato complexes that differ by variations of the substituents on either the phenylpyridine or the tetrazolate ligand have been converted into the corresponding methylated and cationic analogues. NMR (1H and 13C) characterization of the Ir(iii) complexes provided the results in agreement with the chemo- and regioselective character of methylation at the N-3 position of the Ir(iii)-coordinated tetrazolato ring. This evidence was further corroborated by the analysis of the molecular structures of the cationic complexes obtained by X-ray diffraction. In view of the photophysical properties, the addition of a methyl moiety to neutral Ir(iii) tetrazolates, which behave as sky-blue or orange phosphors, caused a systematic red shift of their phosphorescence output. The transformation of neutral Ir(iii) tetrazolates into cationic Ir(iii)-tetrazole complexes was screened for any eventual antimicrobial activity in vitro against Gram negative (E. coli) and Gram positive (D. radiodurans) microorganisms. While both kinds of complexes were not active against E. coli, the conversion of the neutral Ir(iii) tetrazolates into the corresponding methylated and cationic Ir(iii)tetrazole derivatives determined the turn-on of a good to excellent antimicrobial activity toward Gram positive Deinococcus radiodurans, a non-pathogenic bacterium that is listed as one of the toughest microorganisms in light of its outstanding resistance to radiation and oxidative stress.

Effect of Nitrogen Fertilization and Harvest Time on Steviol Glycosides, Flavonoid Composition, and Antioxidant Properties in Stevia rebaudiana Bertoni.

This work investigated the effect of nitrogen fertilization and harvest time on the flavonoid composition and antioxidant properties of Stevia rebaudiana leaves. At the same time, changes in stevioside (Stev) and rebaudioside A (RebA) contents were recorded. A pot trial under open air conditions was set up, testing five N rates and three harvest times. The results showed that, by using an adequate N rate and choosing an appropriate harvest time, it was possible to significantly increase and optimize the bioactive compound levels. In particular, higher RebA, RebA/Stev ratio, total phenols and flavonoids, luteolin-7-O-glucoside, and apigenin-7-O-glucoside levels and antioxidant capacity were recorded by supplying 150 kg N ha(-1). Reduced or increased N availability in comparison with N150 had no consistent effect on Stevia phytochemicals content. Significant correlations were also found between stevioside and some of the flavonoids, indicating a possible role of flavonoids in the stevioside metabolic pathway, which deserves more investigations.

Effects of combined ozone and cadmium stresses on leaf traits in two poplar clones.

Information on plant responses to combined stresses such as ozone (O3) and cadmium (Cd) is scarce in tree species. On the other hand, high O3 concentrations in the atmosphere and heavy metal contaminations in water and soil simultaneously affect forest ecosystems. Toxic metals may exacerbate the consequences of air pollutants. In this research, two poplar clones, differently sensitive to O3 ("I-214" O3-tolerant and "Eridano" O3-sensitive), were grown for 5 weeks in pots supplied with 0 and 150 mg Cd kg(-1) soil and then exposed to a 15-day O3 fumigation (60 nl l(-1), 5 h a day) or supplied with charcoal-filtered air under the same conditions (referred to as control samples). The effects of the two stressors, alone or in combination, on Cd accumulation, photosynthetic capacity, ethylene emission and oxidative state were investigated in fully expanded leaves. Cadmium accumulation in leaves caused a reduction, but not complete failure, of photosynthesis in Eridano and I-214 poplar clones. The reduction in assimilation rate was more important following O3 fumigation. Stomatal aperture after O3 treatment, instead, increased in I-214 and decreased in Eridano. Overall, Cd treatment was effective in decreasing ethylene emission, whereas O3 fumigation increased it in both clones, although interacting with the metal treatment. Again, O3 fumigation induced a significant increase in ascorbate (ASA) + dehydroascorbate (DHA) content, which was strongly oxidised by O3, thus decreasing the redox state. On the other hand, Cd treatment had a positive effect on ASA content and redox state in I-214, but not in Eridano. Although Cd and O3 are known to share some common toxicity pathways, the combined effects induced distinct clone-specific responses, underlying the complexity of plant reactions to multiple stresses.

Targeting divalent metal cations with Re(I) tetrazolato complexes.

In order to exploit their potential as versatile luminescent sensors, four new Re(I)-tetrazolato complexes with the general formula fac-[Re(CO)3(diim)(L)], where diim is 2,2'-bipyridine (bipy) or 1,10-phenanthroline (phen) and L(-) is either the anion 5-(2'-pyridyl)tetrazolato (2-PTZ(-)) or 5-(2'-quinolyl)tetrazolato (2-QTZ(-)), were prepared and fully characterized. In all cases, the regioselective coordination of the Re(I) center through the N2 atom of the tetrazolato ring was observed. This particular feature ensures the availability of the diiminic (N^N) site that was systematically incorporated into the structure of the 2-PTZ(-) and 2-QTZ(-) ligands for further coordination with metal cations. Such a diimine-type coordination mode was preliminarily tested by using the mononuclear Re(I) complexes as N^N ligands for the preparation of two [(N^N)Cu(POP)] cationic species, where POP is the chelating diphosphine bis[2-(diphenylphosphino)phenyl]ether. The X-ray structures of the resulting Re(I)-Cu(I) dyads revealed that the Re(I) mononuclear complexes effectively behaved as chelating N^N ligands with respect to the [Cu(POP)](+) fragment, the coordination of which also resulted in significant modification of the Re(I)-centered luminescence. With these data in hand, the luminescent sensing abilities of the four new Re(I) tetrazolato complexes were screened with respect to divalent metal ions of toxicological and biological importance such as Zn(II), Cd(II) and Cu(II). The interaction of the Re(I) complexes with Zn(II) and Cd(II) was witnessed by the evident blue shift (Δλmax = 22-36 nm) of the emission maxima, which was also accompanied by a significant elongation of the emission lifetimes. On the contrary, the addition of the cupric ion caused substantial quenching of the radiative processes originating from the Re(I) luminophores.

Short-term postharvest carbon dioxide treatments induce selective molecular and metabolic changes in grape berries.

Detached wine grapes ( Vitis vinifera cv. 'Trebbiano', white skinned) were treated for 3 days with 30 kPa of CO(2) and then transferred to air for an additional 9 days to partially dehydrate (about 20% weight loss). At the end of the CO(2) treatment on withering berries, total polyphenols and flavonoids were maintained in the skin, but to a more limited extent in the pulp. An induction of the proanthocyanidin synthesis appeared to be one of the responses to the treatment because both (+)-catechin and (-)-epicatechin concentrations increased in the skin. The skin and pulp of the grape berries showed different molecular responses to a high CO(2) treatment. As revealed by microarray hybridizations, 217 and 75 genes appeared differentially expressed in the skin and pulp of treated samples, respectively. Functional categorization and gene enrichment analyses pointed out that epicarp cells undergo more pronounced changes in transcript profiling at the end of the incubation period. Highly represented categories in both tissues were related to protein, stress, transcript, RNA, and hormone (ethylene, ABA) metabolism. Fermentation, CHO metabolism, and redox regulation functional categories were represented only in the skin.