Tracing amino acid exchange during host-pathogen interaction by combined stable-isotope time-resolved Raman spectral imaging

This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of L-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting L-Phe(D8) from host cells as soon as it invades the cell. L-Phe(D8) from the host cell completely replaces the L-Phe within T. gondii tachyzoites 7–9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5–1.6 × 104 molecules/s. On the other hand, extracellular tachyzoites were not able to consume L-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study

This is the final version. Available on open access from the American Chemical Society via the DOI in this recordThe variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies.COST (European Cooperation in Science and Technology)Portuguese Foundation for Science and TechnologyNational Research Fund of Luxembourg (FNR)China Scholarship Council (CSC)BOKU Core Facilities Multiscale ImagingDeutsche Forschungsgemeinschaft (DFG, German Research Foundation

The Potential of a Novel Concept of an Integrated Bio and Chemical Formulate Based on an Entomopathogenic Bacteria, <i>Bacillus thuringiensis</i>, and a Chemical Insecticide to Control Tomato Leafminer, <i>Tuta absoluta</i> ‘(Meyrick)’ (Lepidoptera: Gelechiidae)

Tomato leafminer, Tuta absoluta (Meyrick), (Lepidoptera: Gelechiidae), poses severe threats to sustainable tomato production globally with a high capacity to develop resistance to pesticides. Recently, the management programs for this cosmopolitan pest have relied on combinations of chemical insecticides which exacerbate the problems of cross-resistance and adverse environmental effects. A novel concept of integrated formulates through combining a chemical insecticide model of lambda-cyhalothrin with Bacillus thuringiensis (Bt) products as the formulation core was explored and evaluated. The susceptibility of the second instar larvae of tomato leafminer to the original formulation of lambda-cyhalothrin, Dipel, XenTari, and Agree products was assessed and compared to the combined formulations. The positive and negative impacts of lambda-cyhalothrin on the viability and pathogenicity of Bt strains were tracked and documented. The physicochemical properties of the combined formulates were examined and compared to the international standards of pesticide formulations. The proposed integrated combined formulates of Dipel, XenTari, and Agree biopesticides with lambda-cyhalothrin showed an enhancing effect and had a higher potential than the originally manufactured formulations alone for about a 3.67–10.08-fold impact on larval mortality. Lambda-cyhalothrin had no significant adverse effect on either the viability of Bt strains or the physicochemical properties of combined co-formulates. Therefore, such integrated combined formulates would have the potential to be involved as an alternative or a complementary approach in pest management and pest resistance management tools for sustainable pest management

Phytochemical and biological assessment of secondary metabolites isolated from a rhizosphere strain, Sphingomonas sanguinis DM of Datura metel

Abstract Background The plant roots excrete a large number of organic compounds into the soil. The rhizosphere, a thin soil zone around the roots, is a hotspot for microbial activity, making it a crucial component of the soil ecosystem. Secondary metabolites produced by rhizospheric Sphingomonas sanguinis DM have sparked significant curiosity in investigating their possible biological impacts. Methods A bacterial strain has been isolated from the rhizosphere of Datura metel. The bacterium’s identification, fermentation, and working up have been outlined. The ethyl acetate fraction of the propagated culture media of Sphingomonas sanguinis DM was fractioned and purified using various chromatographic techniques. The characterization of the isolated compounds was accomplished through the utilization of various spectroscopic techniques, such as UV, MS, 1D, and 2D-NMR. Furthermore, the evaluation of their antimicrobial activity was conducted using the agar well diffusion method, while cytotoxicity was assessed using the MTT test. Results The extract from Sphingomonas sanguinis DM provided two distinct compounds: n-dibutyl phthalic acid (1) and Bis (2-methyl heptyl) phthalate (2) within its ethyl acetate fraction. Furthermore, the 16S rRNA gene sequence of Sphingomonas sanguinis DM has been registered under the NCBI GenBank database with the accession number PP422198. The bacterial extract exhibited its effect against gram-positive bacteria, inhibiting Streptococcus mutans (12.6 ± 0.6 mm) and Staphylococcus aureus (10.6 ± 0.6 mm) compared to standard antibiotics. Conversely, compound 1 showed a considerable effect against phytopathogenic fungi such as Alternaria alternate (56.3 ± 10.6 mm) and Fusarium oxysporum (21.3 ± 1.5 mm) with a MIC value of 17.5 µg/mL. However, it was slightly active against Klebsiella pneumonia (11.0 ± 1.0 mm). Furthermore, compound 2 was the most active metabolite, having a significant antimicrobial efficacy against Rhizoctonia solani (63.6 ± 1.1 mm), Pseudomonas aeruginosa (16.7 ± 0.6 mm), and Alternaria alternate (20.3 ± 0.6 mm) with MIC value at 15 µg/mL. In addition, compound 2 exhibited the most potency against hepatocellular (HepG-2) and skin (A-431) carcinoma cell lines with IC50 values of 107.16 µg/mL and 111.36 µg/mL, respectively. Conclusion Sphingomonas sanguinis DM, a rhizosphere bacterium of Datura metel, was studied for its phytochemical and biological characteristics, resulting in the identification of two compounds with moderate antimicrobial and cytotoxic activities

Raman studies of solution polyglycine conformations

Polyglycine (polygly) is an important model system for understanding the structural preferences of unfolded polypeptides in solution. We utilized UV resonance and visible Raman spectroscopy to investigate the conformational preferences of polygly peptides of different lengths in water containing LiCl and LiClO4. Lithium salts increase the solubility of polygly. Our study indicates that in solution the conformational ensemble of polygly, as well as central peptide bonds of gly5 and gly6, are dominated by the 31 extended helix, also known as the polyglycine II conformation (PGII). This preference of the polygly backbone for the PGII conformation in solution is likely a result of favorable interactions between carbonyl dipoles in these extended helices. We found that high concentrations of Li+ stabilize the PGII conformation in solution, most likely by polarizing the peptide bond carbonyls that makes PGII-stabilizing carbonyl-carbonyl electrostatic interactions more favorable. This ability of Li+ to stabilize 31-helix conformations in solution gives use to the denaturing ability of lithium salts. © 2010 American Chemical Society

Spectral Pathology: general discussion.

Parvez Haris opened the discussion of the introductory lecture by Max Diem: Varying degrees of accuracy have been obtained for discrimination between cancerous and non-cancerous tissues using vibrational spectroscopic methods. What are the explanations for these variation in accuracy between cancerous and non-cancerous tissues and how do they correlate with accuracy from other techniques including histopathology