Bioimaging
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Chemické transformace kompatibilní s biologickými systémy slouží jako neocenitelné nástroje pro zkoumání biomolekul, sloučenin léčiv a biologických procesů v jejich přirozeném prostředí. Složité prostředí živých organismů vyžaduje, aby tyto reakce byly vysoce selektivní a účinné, což představuje pro oblast organické chemie obrovskou výzvu. V poslední době se objevila řada chemických reakcí, které tyto podmínky splňují, a poskytují proto výzkumným pracovníkům rozmanitou sadu nástrojů. Tento rukopis představuje komplexní přehled současných bioortogonálních reakcí s důrazem na jejich aplikace v zobrazování, diagnostice a medicíně.
Chemical transformations compatible with biological systems serve as invaluable tools for probing biomolecules, drug compounds, and biological processes in their native environments. The complex environment of living organisms requires these reactions to be highly selective and efficient, posing a formidable challenge to the field of organic chemistry. A number of chemical reactions have recently emerged to meet this challenge, providing a diverse toolkit for researchers. This manuscript presents a comprehensive survey of current bioorthogonal reactions, emphasizing their applications in bioimaging, diagnostics, and medicine.
Identifikace buněčných cílů aktivních látek má zásadní význam pro optimalizaci léčiv a minimalizaci jejich nežádoucích vedlejších účinků. Komplexní povaha biologických systémů ztěžuje tuto identifikaci, ale mikroskopické metody, zejména fenotypové testování, reprezentované metodou „Cell Painting“, představují cenný nástroj pro pochopení vlivu látek na úrovni buněk a organel. Tyto metody umožňují rychlé testování rozsáhlých knihoven látek a nabízejí unikátní pohled na mechanismus jejich účinku pozorováním chování buněk, pomocí hodnocení jejich morfologie, pohyblivosti, dělení a migrace. Mikroskopie živých buněk čelí výzvám, jako je fototoxicita, což vyžaduje pečlivý výběr fluorescenčních značek a optimalizaci podmínek. Mezi syntetickými fluorescenčními sondami pro mikroskopii živých buněk vynikají BODIPY barviva se svou syntetickou univerzálností a fotofyzikálními vlastnostmi, které zajišťují minimální poškození vzorku během biozobrazování.
Target identification of active substances is critical in optimizing drugs and minimizing side effects. The complex nature of biological systems presents challenges; to meet them, however, microscopic methods, particularly phenotypic screening, represented by "Cell Painting" method and fluorescent probes, can be used as valuable tools for understanding the impact of various substances at the cellular and organelle levels. These methods enable rapid testing of large libraries of compounds and offer unique insights into their mechanism of action by observing cell behavior, assessing cell morphology, motility, division, and migration. However, live cell microscopy faces challenges like phototoxicity, requiring a careful selection of fluorescent labels and optimized conditions. Among synthetic probes for live cell microscopy, BODIPY dyes stand out for their synthetic versatility and photophysical properties, providing minimal sample damage during bioimaging.
BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) is well-established in neuronal function, yet its role in immune reactions remains enigmatic. The conflicting data on its inflammatory role, suggesting both pro-inflammatory and anti-inflammatory effects upon TRPV1 stimulation in immune cells, adds complexity. To unravel TRPV1 immunomodulatory mechanisms, we investigated how the TRPV1 agonist capsaicin influences lipopolysaccharide (LPS)-induced pro-inflammatory macrophage phenotypes. RESULTS: Changes in the surface molecules, cytokine production, and signaling cascades linked to the phenotype of M1 or M2 macrophages of the J774 macrophage cell line and bone marrow-derived macrophages, treated with capsaicin before or after the LPS-induced inflammatory reaction were determined. The functional capacity of macrophages was also assessed by infecting the stimulated macrophages with the intracellular parasite Leishmania mexicana. CONCLUSION: Our findings reveal that TRPV1 activation yields distinct macrophage responses influenced by the inflammatory context. LPS pre-treatment followed by capsaicin activation prompted increased calcium influx, accompanied by a shift toward an anti-inflammatory M2b-like polarization state.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The prognostic significance of mast cells and different phenotypes of macrophages in the microenvironment of hepatocellular carcinoma (HCC) following resection is unclear. We aimed in this study to assess the local distribution of infiltrating macrophages and mast cells of specific phenotypes in tissues of HCC and to evaluate their prognostic values for survival of post-surgical patients. METHODS: The clinicopathological and follow-up data of 70 patients with HCC, who underwent curative resection of tumor from 1997 to 2019, were collected. The infiltration of CD68+ and CD163+ macrophages and CD117+ mast cells was assessed immunohistochemically in representative resected specimens of HCC and adjacent tissues. The area fraction (AF) of positively stained cells was estimated automatically using QuPath image analysis software in several regions, such as tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells, individually and in associations, for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) was evaluated using Kaplan-Meier and Cox regression analyses. RESULTS: High AF of CD68+ macrophages in TC and IM and high AF of mast cells in IM and PT area were associated with a longer DFS. High AF of CD163+ macrophages in PT area correlated with a shorter DFS. Patients from CD163TChigh & CD68TClow group had a shorter DFS compared to all the rest of the groups, and cases with CD163IMlow & CD68IMhigh demonstrated significantly longer DFS compared to low AF of both markers. Patients from CD68IMhigh & CD163PTlow group, CD117IMhigh & CD163PTlow group, and CD117PThigh & CD163PTlow group had a significantly longer DFS compared to all other combinations of respective cells. CONCLUSIONS: The individual prognostic impact of CD68+ and CD163+ macrophages and mast cells in the microenvironment of HCC after resection depends on their abundance and location, whereas the cumulative impact is built upon combination of different cell phenotypes within and between regions.
PURPOSE: Water removal is one of the computational bottlenecks in the processing of high-resolution MRSI data. The purpose of this work is to propose an approach to reduce the computing time required for water removal in large MRS data. METHODS: In this work, we describe a singular value decomposition-based approach that uses the partial position-time separability and the time-domain linear predictability of MRSI data to reduce the computational time required for water removal. Our approach arranges MRS signals in a Casorati matrix form, applies low-rank approximations utilizing singular value decomposition, removes residual water from the most prominent left-singular vectors, and finally reconstructs the water-free matrix using the processed left-singular vectors. RESULTS: We have demonstrated the effectiveness of our proposed algorithm for water removal using both simulated and in vivo data. The proposed algorithm encompasses a pip-installable tool ( https://pypi.org/project/CSVD/), available on GitHub ( https://github.com/amirshamaei/CSVD), empowering researchers to use it in future studies. Additionally, to further promote transparency and reproducibility, we provide comprehensive code for result replication. CONCLUSIONS: The findings of this study suggest that the proposed method is a promising alternative to existing water removal methods due to its low processing time and good performance in removing water signals.
(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.
Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to mediate cellular processes such as mitosis and intracellular transport. In trypanosomatids, the kinesin superfamily has undergone a prominent expansion, resulting in one of the most diverse kinesin repertoires that includes the two kinetoplastid-restricted families X1 and X2. Here, we characterize in Trypanosoma brucei TbKifX2A, an orphaned X2 kinesin. TbKifX2A tightly interacts with TbPH1, a kinesin-like protein with a likely inactive motor domain, a rarely reported occurrence. Both TbKifX2A and TbPH1 localize to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that wraps around the flagellar pocket as it extends to the cell body anterior. The proximal proteome of TbPH1 revealed two other interacting proteins, the flagellar pocket protein FP45 and intriguingly another X2 kinesin, TbKifX2C. Simultaneous ablation of TbKifX2A/TbPH1 results in the depletion of FP45 and TbKifX2C and also an expansion of the flagellar pocket, among other morphological defects. TbKifX2A is the first motor protein to be localized to the MtQ. The observation that TbKifX2C also associates with the MtQ suggests that the X2 kinesin family may have co-evolved with the MtQ, both kinetoplastid-specific traits.
This study illustrates the synthesis of functionalized carbon quantum dots (CQDs) by the one-pot pyrolysis method. The functionalization agent used in CQD synthesis was poly l- lysine (PLL). Various physicochemical techniques were employed to confirm the successful formation of PLLCQD including High resolution transmission electron microscopy (HR-TEM), UV-Vis spectroscopy, fluorescence spectroscopy; Atomic force microscopy (AFM), X-ray Photoelectron Spectroscopy (XPS) and X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The size of PLLCQD was confirmed by HRTEM and AFM. The synthesized PLLCQD shows bright blue fluorescence and has a quantum yield of 19.35%. The highest emission band was observed at 471nm when excited to 370nm. The prepared PLLCQD exhibited excellent antibacterial activity against Escherichia coli and Staphylococcus aureus with inhibition zone 7-20 mm. The concentrations of 0.9 to 0.1gmL-1 were studied to determine minimum inhibitory concentration (MIC) by the agar well diffusion assay method. MIC of 0.2gml -1 concentration of PLLCQD is achieved. The anti-angiogenic activity of PLLCQD was determined using (Chick Chorioallantoic Membrane) CAM assay. CAM assay is a reliable in -vivo model to study angiogenesis also; many stimulators and inhibitors have been examined by this method. This study proves higher antibacterial efficiency of PLLCQD over non functionalized CQD. PLLCQD was successfully employed in bio-imaging of the bacterial cell through fluorescence microscopy. Further, PLLCQD displayed cytotoxic effect on endothelial cells and inhibited blood vessel formation in the CAM model.
Early postnatal events are important for the development of the neonatal immune system. Harboring the pioneering microorganisms forming the microbiota of the neonatal gastrointestinal tract is important for priming the immune system, as well as inducing appropriate tolerance to the relatively innocuous environmental antigens and compounds of normal healthy microbiota. Early postnatal supplementation of suitable, safe probiotics could accelerate this process. In the current study, the immunomodulatory capacity of the probiotic strain of Escherichia coli O83:K24:H31 (EcO83) was characterized in vitro and in vivo. We compared the capacity of EcO83 with and without hemolytic activity on selected immune characteristics in vitro as determined by flow cytometry and quantitative real-time PCR. Both strains with and without hemolytic activity exerted comparable capacity on the maturation of dendritic cells while preserving the induction of interleukin 10 (Il10) expression in dendritic cells and T cells cocultured with EcO83 primed dendritic cells. Early postnatal supplementation with EcO83 led to massive but transient colonization of the neonatal gastrointestinal tract, as detected by in vivo bioimaging. Early postnatal EcO83 administration promoted gut barrier function by increasing the expression of claudin and occludin and the expression of Il10. Early postnatal EcO83 application promotes maturation of the neonatal immune system and promotes immunoregulatory and gut barrier functions.
- MeSH
- dendritické buňky MeSH
- Escherichia coli MeSH
- interleukin-10 MeSH
- lidé MeSH
- mikrobiota * MeSH
- novorozenec MeSH
- probiotika * farmakologie MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
