A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism.
Ontology highlight
ABSTRACT:
SUBMITTER: de Moliner F
PROVIDER: S-EPMC8048481 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:We propose to monitor molecular vibrations to identify metal ion-ligand complexation by means of Raman spectroscopy, which has been applied to track vibrational modes of molecules and to obtain a structural fingerprint. We prepared ligand molecules for Zn2+ ion complexation with a dipycolylaminoethyl aniline (DPEA) skeleton and phenylacetylene unit as the Raman tag which showed a typical band around 2200 cm-1. Among the labeled ligands synthesized in this study, A-DPEA showed a strong band attributed to the acetylene unit at 2212 cm-1, while the addition of Zn2+ ion resulted in a band shift to 2220 cm-1 due to complex formation. The addition of other metal ions and titration experiments showed that A-DPEA bound with Zn2+ selectively with a dissociation constant (K d) that was estimated to be 0.22 μM. We also conducted cellular experiments and found that complexation between A-DPEA and Zn2+ also occurred in cells, with a shift in the Raman signal of the ligand from 2212 to 2215 cm-1. Thus, complex formation of the metal ion was identified by monitoring the Raman band shift.
| S-EPMC9056985 | biostudies-literature
Project description:Antibiotics resistance developed by biofilms has posed a clinical challenge in the effective treatment of bacterial infections. However, the resistance mechanisms have not been well understood due to a lack of suitable tools for dynamic observation of the interplay between antibiotics and biofilm. In this work, with the use of rapid hyperspectral stimulated Raman scattering microscopy associated with an aryl-alkyne-based Raman tag synthesized, we investigate dynamic interactions between vancomycin and Staphylococcus aureus (S. aureus) biofilm to gain new insights into the resistance mechanisms of the biofilm. Methods: We utilize spectral focusing hyperspectral stimulated Raman scattering microscopy ensued with multivariate curve resolution analysis to spectrally decompose S. aureus biofilm into its major components (i.e., bacteria and extracellular polymeric substances). Concurrently, vancomycin is conjugated with aryl-alkyne Raman tag (Raman peak at 2218 cm-1) for in vivo tracking of its uptake into biofilm without tissue interference. Results: We find that vancomycin penetration is a non-uniform diffusion process with penetration depths limited by the preferential affinity to the cell clusters. Semi-quantitative analysis shows that the majority of vancomycin binds to the bacteria, achieving intracellular concentrations of up to 4- to 10- fold higher than the administered dosage. The diffusion constant of ~3.16 µm2/min based on the diffusion and antibiotic binding equations is obtained that well accounts for the antibiotic penetration into the biofilm. SRS longitudinal monitoring of antibiotic effect on the growth of biofilms shows that the antibiotics can eradicate the upper layer of the biofilm exposed to sufficient dosages, while the lower layer of the biofilm at a sub-inhibitory dose remains viable, eventually re-growing to significant bio-volume. Conclusion: The Raman-tagged hyperspectral SRS microscopy developed is a powerful imaging tool for dynamic monitoring of inhibitory effects of antibiotics on the growing biofilm in vivo, which would facilitate the formulation of new antibiotics for more effective treatments of bacterial infections in near future.
| S-EPMC6401501 | biostudies-literature
Project description:Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species. This indicates the promise of using Raman spectroscopy directly with alkyne tagged lipids for cellular studies as opposed to subsequently clicking of a fluorophore onto the alkyne for fluorescence imaging.
| S-EPMC5890826 | biostudies-other
Project description:Streptococcus mutans converts extracellular sucrose (Suc) into exopolysaccharides (EPS) by glucosyl-transferase and fructosyl-transferase enzymes and internalizes Suc for fermentation through the phosphotransferase system (PTS). Here, we examined how altering the routes for sucrose utilization impacts intracellular polysaccharide [IPS; glycogen, (glg)] metabolism during carbohydrate starvation. Strain UA159 (WT), a mutant lacking all exo-enzymes for sucrose utilization (MMZ952), and a CcpA-deficient mutant (∆ccpA) were cultured with sucrose or a combination of glucose and fructose, followed by carbohydrate starvation. At baseline (0h), and after 4 and 24h of starvation, cells were evaluated for mRNA levels of the glg operon, IPS storage, glucose-1-phosphate (G1P) concentrations, viability, and PTS activities. A pH drop assay was performed in the absence of carbohydrates at the baseline to measure acid production. We observed glg operon activation in response to starvation (p<0.05) in all strains, however, such activation was significantly delayed and reduced in magnitude when EPS synthesis was involved (p<0.05). Enhanced acidification and greater G1P concentrations were observed in the sucrose-treated group, but mostly in strains capable of producing EPS (p<0.05). Importantly, only the WT exposed to sucrose was able to synthesize IPS during starvation. Contrary to CcpA-proficient strains, IPS was progressively degraded during starvation in ∆ccpA, which also showed increased glg operon expression and greater PTS activities at baseline. Therefore, sucrose metabolism by secreted enzymes affects the capacity of S. mutans in synthesizing IPS and converting it into organic acids, without necessarily inducing greater expression of the glg operon.
| S-EPMC7884614 | biostudies-literature
Project description:Fluorescence live imaging has become an essential methodology in modern cell biology. However, fluorescence requires excitation light, which can sometimes cause potential problems, such as autofluorescence, phototoxicity, and photobleaching. Furthermore, combined with recent optogenetic tools, the light illumination can trigger their unintended activation. Because luminescence imaging does not require excitation light, it is a good candidate as an alternative imaging modality to circumvent these problems. The application of luminescence imaging, however, has been limited by the two drawbacks of existing luminescent protein probes, such as luciferases: namely, low brightness and poor color variants. Here, we report the development of bright cyan and orange luminescent proteins by extending our previous development of the bright yellowish-green luminescent protein Nano-lantern. The color change and the enhancement of brightness were both achieved by bioluminescence resonance energy transfer (BRET) from enhanced Renilla luciferase to a fluorescent protein. The brightness of these cyan and orange Nano-lanterns was ?20 times brighter than wild-type Renilla luciferase, which allowed us to perform multicolor live imaging of intracellular submicron structures. The rapid dynamics of endosomes and peroxisomes were visualized at around 1-s temporal resolution, and the slow dynamics of focal adhesions were continuously imaged for longer than a few hours without photobleaching or photodamage. In addition, we extended the application of these multicolor Nano-lanterns to simultaneous monitoring of multiple gene expression or Ca(2+) dynamics in different cellular compartments in a single cell.
| S-EPMC4394297 | biostudies-literature
Project description:Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H(+) symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level.
| S-EPMC3071760 | biostudies-literature
Project description:ObjectivesTo evaluate a novel method for real-time tagged MRI with increased tag persistence using phase sensitive tagging (REALTAG), demonstrated for speech imaging.MethodsTagging is applied as a brief interruption to a continuous real-time spiral acquisition. REALTAG is implemented using a total tagging flip angle of 180° and a novel frame-by-frame phase sensitive reconstruction to remove smooth background phase while preserving the sign of the tag lines. Tag contrast-to-noise ratio of REALTAG and conventional tagging (total flip angle of 90°) is simulated and evaluated in vivo. The ability to extend tag persistence is tested during the production of vowel-to-vowel transitions by American English speakers.ResultsREALTAG resulted in a doubling of contrast-to-noise ratio at each time point and increased tag persistence by more than 1.9-fold. The tag persistence was 1150 ms with contrast-to-noise ratio >6 at 1.5T, providing 2 mm in-plane resolution, 179 frames/s, with 72.6 ms temporal window width, and phase sensitive reconstruction. The new imaging window is able to capture internal tongue deformation over word-to-word transitions in natural speech production.ConclusionTag persistence is substantially increased in intermittently tagged real-time MRI by using the improved REALTAG method. This makes it possible to capture longer motion patterns in the tongue, such as cross-word vowel-to-vowel transitions, and provides a powerful new window to study tongue biomechanics.
| S-EPMC7180094 | biostudies-literature
Project description:There is considerable interest in the use of sugars to preserve cells. In this study, low temperature Raman spectroscopy was used to characterize the behaviors of sucrose during freezing. The hydrogen bond network between sucrose and water was investigated at -10 °C and -50 °C, and the Raman spectra showed strengthened sucrose-water and sucrose-sucrose hydrogen bonds in more concentrated sucrose solution at -50 °C. The concentration of sucrose at the ice interface increased as the ice density decreased, and it plateaued across a narrow channel of nonfrozen sucrose solution before it decreased toward the next ice interface. The biophysical environment at interfaces between the cell and nonfrozen sucrose solution and between the cell and extracellular ice was also studied. A thin layer of nonfrozen sucrose solution was observed at the interface between the cell and extracellular ice. The extracellular concentration of sucrose at this interface was generally lower than that of bulk nonfrozen sucrose solution. The variation of sucrose concentration outside different regions of the cell membrane suggests that the chemical environment around the cell during freezing may be more heterogeneous than previously thought. Raman spectra and images also showed colocalization of nonfrozen sucrose solution and the cell, which implied that direct interaction between sucrose and cell membrane might be responsible for protective properties of sucrose. Sucrose was predominantly distributed outside the cell, and the observation of strong partitioning of sucrose across the cell membrane is consistent with substantial cell dehydration detected by the Raman spectra. This work enhances our understanding of the behaviors of sucrose solution and its interactions with cells at low temperature and can improve cryopreservation protocols of cells frozen in a sucrose-based media.
| S-EPMC8023323 | biostudies-literature
Project description:Cholesterol is an important lipid playing a crucial role in mediating essential cellular processes as well as maintaining the basic structural integrity of biological membranes. Given its vast biological importance, there is an unabated need for sophisticated strategies to investigate cholesterol-mediated biological processes. Raman-tagged sterol analogs offer the advantage of being visualizable without the need for a bulky dye that potentially affects natural membrane integration and cellular interactions as it is the case for many conventionally used fluorescent analogs. Herein, we report a series of alkyne-tagged imidazolium-based cholesterol analogs (CHIMs) with large Raman scattering cross-sections that readily integrate into HEK cells and primary monocyte-derived macrophages and allow (multiplexed) cellular Raman imaging. We envision Raman-tagged CHIM analogs to be a powerful platform for the investigation of cholesterol-mediated cellular processes complementary to other established methods, such as the use of fluorescent analogs.
| S-EPMC11325193 | biostudies-literature
Project description:Sensitive and specific visualization of small biomolecules in living systems is highly challenging. We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors.
| S-EPMC4040164 | biostudies-literature