The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self-assembly of hIAPP22–29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe-23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman, and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe-23 virtually abolish the ability of hIAPP22–29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β-sheet secondary structure. TEM images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the -C=C- ring mode (ca. 1600 cm−1) upon aggregation that has previously been shown to be indicative of π-stacking. While previous work has demonstrated that π-stacking is not an absolute requirement for fibrillization, our findings indicate that Phe-23 also contributes to fibril formation through π-stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self-assembly of hIAPP22–29.
The unforeseen COVID-19 pandemic forced educational institutions to shift to a remote or distance-learning mode. As a result, classes were offered online, and this shift in teaching modality presented great challenges, especially in teaching laboratory courses. While several options are available, we evaluated the use of (i) videos of lab demonstrations, (ii) Microsoft PowerPoint slides with voice-over recordings that were prepared to guide students further in the particular procedure of the experiment, and (iii) kitchen-based experiments that students could perform at home for our General Chemistry I laboratory course that was offered in an asynchronous modality during the Summer session. The students were surveyed for feedback, comments, and reactions to the use of these different practices. On the basis of student comments, it was found that the videos were beneficial to illustrate important aspects of each experiment, with some students commenting that it made them feel as if they were actually performing the experiments themselves. The kitchen-based experiments, on the other hand, allowed students to experience performing hands-on experiments and helped them observe and relate to concepts (such as classifying matter, making physical measurements, employing units and significant figures, preparing solutions, calculating moles and molarity, and employing separation techniques) that were discussed in the lecture portion of the course.
Tetracycline antibiotics, such as chlortetracycline (CTC) and tetracycline (TC), are introduced into agricultural lands through the application of manure as fertilizer. These compounds are phytotoxic to certain crop plants, including pinto beans (Phaseolus vulgaris), the species used for this investigation. While the mechanism of this toxicity is not yet understood, CTC is known to be a calcium chelator. We describe here a novel method to show that CTC is taken up by pinto bean plants and chelates calcium in leaves. Cameleon fusion proteins can provide qualitative and quantitative imaging of intracellular calcium levels, but current methodology requires stable transformation. Many plant species, including pinto beans, are not yet transformable using standard Agrobacterium-based protocols. To determine the role of calcium chelation in this plant, a rapid, biolistic method was developed to transiently express the cameleon protein. This method can easily be adapted to other plant systems. Our findings provide evidence that chelation of intracellular calcium by CTC is related to phytotoxic effects caused by this antibiotic in pinto beans. Root uptake of CTC and TC by pinto beans and their translocation to leaves were further verified by fluorescence spectroscopy and liquid chromatography/mass spectrometry, confirming results of the biolistic method that showed calcium chelation by tetracyclines in leaves.
The creation of tetracycline (TC) responsive molecularly imprinted xerogels (MIXs) was investigated using electronic absorbance, liquid chromatography-ion-trap mass spectrometry (LC-ITMS), and first-principles theory. Experimental results show that the template molecule converts to its epimer, 4-epitetracycline (ETC), during the imprinting process. Additionally, end capping of the MIX surface silanols transforms TC into anhydrotetracycline (ATC) and 4-epianhydrotetracycline (EATC). Hence, despite aiming to imprint for a single analyte (TC), one simultaneously imprints for up to four analogs (TC, ETC, EATC and ATC) within a MIX. Binding studies using LC-MS showed the binding of the prepared xerogels with the four analogs. In some formulations, preferential uptake of ETC, EATC and ATC relative to the template molecule (TC) was observed. Computations of the interaction energies between silane monomers and the four analogs reveal that ETC, EATC and ATC have higher interaction energies and are more likely to be imprinted in comparison to TC.
Plant responses to natural stresses have been the focus of numerous studies; however less is known about plant responses to artificial (i.e., man-made) stress. Chlortetracycline (CTC) is widely used in agriculture and becomes an environmental contaminant when introduced into soil from manure used as fertilizer. We show here that in the model plant Arabidopsis (Arabidopsis thaliana), root uptake of CTC leads to toxicity, with growth reductions and other effects. Analysis of protein accumulation and in vivo synthesis revealed numerous changes in soluble and membrane-associated proteins in leaves and roots. Many representative proteins associated with different cellular processes and compartments showed little or no change in response to CTC. However, differences in accumulation and synthesis of NAD-malic enzyme in leaves versus roots suggest potential CTC-associated effects on metabolic respiration may vary in different tissues. Fluorescence resonance energy transfer (FRET) analysis indicated reduced levels of intracellular calcium are associated with CTC uptake and toxicity. These findings support a model in which CTC uptake through roots leads to reductions in levels of intracellular calcium due to chelation. In turn, changes in overall patterns and levels of protein synthesis and accumulation due to reduced calcium ultimately lead to growth reductions and other toxicity effects.
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