Quantitative colocalization analysis is an advanced digital imaging tool to examine antigens of interest in immunofluorescence images obtained using confocal microscopes. It employs specialized ...algorithms to estimate the degree of overlap of fluorescence signals and thus enables acquiring important new information not otherwise obtainable using qualitative approaches alone. As raw confocal images have high levels of background, they should be prepared to become suitable for reliable calculation of colocalization coefficients by correcting it. We provide concise theoretical basis of quantitative colocalization analysis, discuss its limitations, and describe proper use of the technique. The use of quantitative colocalization analysis is demonstrated by studying bile salt export pump and multidrug resistance associated protein 2 in the liver and major basic protein and platelet activating factor receptor antigens in conjunctiva. The review is focused on the applicability and correct interpretation of the results of colocalization coefficients calculations.
Genome-wide association studies (GWAS) have successfully mapped thousands of loci associated with complex traits. These associations could reveal the molecular mechanisms altered in common complex ...diseases and result in the identification of novel drug targets. However, GWAS have also left a number of outstanding questions. In particular, the majority of disease-associated loci lie in non-coding regions of the genome and, even though they are thought to play a role in gene expression regulation, it is unclear which genes they regulate and in which cell types or physiological contexts this regulation occurs. This has hindered the translation of GWAS findings into clinical interventions. In this review we summarize how these challenges have been addressed over the last decade, with a particular focus on the integration of GWAS results with functional genomics datasets. Firstly, we investigate how the tissues and cell types involved in diseases can be identified using methods that test for enrichment of GWAS variants in genomic annotations. Secondly, we explore how to find the genes regulated by GWAS loci using methods that test for colocalization of GWAS signals with molecular phenotypes such as quantitative trait loci (QTLs). Finally, we highlight potential future research avenues such as integrating GWAS results with single-cell sequencing read-outs, designing functionally informed polygenic risk scores (PRS), and validating disease associated genes using genetic engineering. These tools will be crucial to identify new drug targets for common complex diseases.
Background: Identifying effective pharmacological interventions to prevent the progressive enlargement and rupture of aortic aneurysms (AAs) is critical. Previous studies have suggested links between ...metformin use and a decreased incidence of AAs. In this study, we employed Mendelian randomization (MR) to investigate causal effects of metformin’s targets on AA risk and to explore the underlying mechanisms underlying these effects. Methods: To examine the relationship between metformin use and AA risk, we implemented both two-sample MR and multivariable MR analyses. Utilizing genetic instrumental variables, we retrieved cis-expression quantitative trait loci (cis-eQTL) data for potential targets of metformin from the Expression Quantitative Trait Loci Genetics Consortium (eQTLGen) Consortium and Genotype-Tissue Expression (GTEx) project. Colocalization analysis was employed to ascertain the probability of shared causal genetic variants between single nucleotide polymorphisms (SNPs) associated with eQTLs and AA. Results: Our findings reveal that metformin use reduces AA risk, exhibiting a protective effect with an odds ratio (OR) of 4.88 × 10-3 (95% confidence interval CI: 7.30 × 10-5–0.33, p = 0.01). Furthermore, the protective effect of type 2 diabetes on AA risk appears to be driven by metformin use (ORMVMR = 1.34 × 10-4, 95% CI: 3.97 × 10-8–0.45, p = 0.03). Significant Mendelian randomization (MR) results were observed for the expression of two metformin-related genes in the bloodstream: NADH:ubiquinone oxidoreductase subunit A6 (NDUFA6) and cytochrome b5 type B (CYB5B), across two independent datasets (ORCYB5B = 1.35, 95% CI: 1.20–1.51, p = 2.41 × 10-7; ORNDUFA6 = 1.12; 95% CI: 1.07–1.17, p = 1.69 × 10-6). The MR analysis of tissue-specific expression also demonstrated a positive correlation between increased NDUFA6 expression and heightened AA risk. Lastly, NDUFA6 exhibited evidence of colocalization with AA. Conclusions: Our study suggests that metformin may play a significant role in lowering the risk of AA. This protective effect could potentially be linked to the mitigation of mitochondrial and immune dysfunction. Overall, NDUFA6 has emerged as a potential mechanism through which metformin intervention may confer AA protection.
Intracellular vesicle trafficking involves a complex series of biological pathways used to sort, recycle, and degrade extracellular components, including engineered nanomaterials (ENMs) which gain ...cellular entry via active endocytic processes. A recent emphasis on routes of ENM uptake has established key physicochemical properties which direct certain mechanisms, yet relatively few studies have identified their effect on intracellular trafficking processes past entry and initial subcellular localization. Here, we developed and applied an approach where single-walled carbon nanotubes (SWCNTs) play a dual rolethat of an ENM undergoing intracellular processing, in addition to functioning as the signal transduction element reporting these events in individual cells with single organelle resolution. We used the exceptional optical properties exhibited by noncovalent hybrids of single-stranded DNA and SWCNTs (DNA–SWCNTs) to report the progression of intracellular processing events via two orthogonal hyperspectral imaging approaches of near-infrared (NIR) fluorescence and resonance Raman scattering. A positive correlation between fluorescence and G-band intensities was uncovered within single cells, while exciton energy transfer and eventual aggregation of DNA–SWCNTs were observed to scale with increasing time after internalization. An analysis pipeline was developed to colocalize and deconvolute the fluorescence and Raman spectra of subcellular regions of interest (ROIs), allowing for single-chirality component spectra to be obtained with submicron spatial resolution. This approach uncovered correlations between DNA–SWCNT concentration, dielectric modulation, and irreversible aggregation within single intracellular vesicles. An immunofluorescence assay was designed to directly observe the DNA–SWCNTs in labeled endosomal vesicles, revealing a distinct relationship between the physical state of organelle-bound DNA–SWCNTs and the dynamic luminal conditions during endosomal maturation processes. Finally, we trained a machine learning algorithm to predict endosome type using the Raman spectra of the vesicle-bound DNA–SWCNTs, enabling major components in the endocytic pathway to be simultaneously visualized using a single intracellular reporter.
Genome-wide association studies (GWAS) have revealed numerous loci associated with multiple sclerosis (MS). However, the challenge lies in deciphering the mechanisms by which these loci influence the ...target traits. Here, we employed an integrative analytical pipeline to efficiently transform genetic associations to identify novel proteins for MS.
We systematically integrated MS GWAS data (N = 115,803) with human plasma proteome data (N = 7213) and conducted proteome-wide association studies (PWAS) to identify MS-associated pathogenic proteins. Following this, we employed Mendelian randomization and Bayesian colocalization analyses to verify the causal relationship between these significant plasma proteins and MS. Lastly, we utilized the Drug-Gene Interaction Database (DGIdb) to identify potential drug targets for MS.
The PWAS identified 25 statistically significant cis-regulated plasma proteins associated with MS at a false discovery rate of P < 0.05. Further analysis revealed that the abundance of 7 of these proteins (PLEK, TNXB, CASP3, CD59, CR1, TAPBPL, ATXN3) was causally related to the incidence of MS. Our findings indicated that genetically predicted higher levels of TNXB and CD59 were associated with a lower risk of MS, whereas higher levels of PLEK, CASP3, CR1, TAPBPL, and ATXN3 were associated with an increased risk of MS. Three plasma proteins (PLEK, CR1, CD59) were validated by colocalization analysis. Among these, CR1 was prioritized as a target for Eculizumab due to its significant association with MS risk. Additionally, PLEK, CR1, and CD59 were identified as druggable target genes.
Our proteomic analysis has identified PLEK, CR1, and CD59 as potential drug targets for MS treatment. Developing pharmacological inducers or inhibitors for these proteins could pave the way for new therapeutic approaches, potentially improving outcomes for MS patients.
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•GWAS has found many MS risk loci, but deciphering the mechanisms is challenging.•We employed an integrative analytical pipeline to identify novel plasma proteins for MS.•Three proteins were identified as compelling proteins (i.e., PLEK, CR1, and CD59).•These proteins may offer insights for MS research and potential diagnostic targets.
Several fluorescence and colorimetric chemosensory for Sn2+ detection in an aqueous media have been reported, but applications remain limited for discriminative Sn2+ detection in live human cells and ...zebrafish larvae. Herein, a mitochondria-targeted Sn2+ “turn-on” colorimetric and fluorescence chemosensor, 2CTA, with an aggregation-induced emission (AIE) response was developed. The sensing of Sn2+ was enabled by a reduction-enabled binding pathway, with the conversion of –C˭O groups to –C–OH groups at the naphthoquinone moiety. The color changed from light maroon to milky white in a buffered aqueous solution. The chemosensor 2CTA possessed the excellent characteristics of good water solubility, fast response (less than 10 s), and high sensitivity (79 nM) and selectivity for Sn2+ over other metal ions, amino acids, and peptides. The proposed binding mechanism was experimentally verified by means of FT-IR and NMR studies. The chemosensor 2CTA was successfully employed to recognize Sn2+ in live human cells and in zebrafish larvae. In addition, a colocalization study proved that the chemosensor had the ability to target mitochondria and overlapped almost completely with MitoTracker Red. Furthermore, a bioimaging study of live cells demonstrated the discriminative detection of Sn2+ in human cancer cells and the practical applications of 2CTA in biological systems.
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•A mitochondria-targeted colorimetric/fluorescence chemosensor 2CTA was developed.•2CTA showed excellent sensitivity to Sn2+ at a nanomolar level (79 nM).•2CTA discriminatively detected Sn2+ in cancer cells than the normal live cells.•The probe was used as a mitochondria-targeted fluorescent marker.•The probe was used for the tracking of Sn2+ in live cells and zebrafish.
Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes ...assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.
Defect engineering is a strategy that has been widely used to design active semiconductor photocatalysts. However, understanding the role of defects, such as oxygen vacancies, in controlling ...photocatalytic activity remains a challenge. Here, we report the use of chemically triggered fluorogenic probes to study the spatial distribution of active regions in individual tungsten oxide nanowires using super-resolution fluorescence microscopy. The nanowires show significant heterogeneity along their lengths for the photocatalytic generation of hydroxyl radicals. Through quantitative, coordinate-based colocalization of multiple probe molecules activated by the same nanowires, we demonstrate that the nanoscale regions most active for the photocatalytic generation of hydroxyl radicals also possess a greater concentration of oxygen vacancies. Chemical modifications to remove or block access to surface oxygen vacancies, supported by calculations of binding energies of adsorbates to different surface sites on tungsten oxide, show how these defects control catalytic activity at both the ensemble and single-particle levels. These findings reveal that clusters of oxygen vacancies activate surface-adsorbed water molecules toward photo-oxidation to produce hydroxyl radicals, a critical intermediate in several photocatalytic reactions.
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•Liposomes modified by OH- and imidazolium-containing amphiphiles have been fabricated.•Its stability during the storage is more than 6-fold higher, than unmodified ...ones.•Chloramphenicol and cisplatin have been encapsulated into fabricated vehicles.•Properties of liposomes could be regulated by selecting amphiphile amount.•Its cytotoxicity against cancer cells and reduced hemolytic activity have been shown.
In the framework of this investigation novel modified liposomal formulations based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and hydroxyethylated imidazolium-containing amphiphiles with various length of hydrophobic tail have been fabricated and characterized by various physicochemical methods. Variation of amphiphile/lipid molar ratio has been used as a tool for optimization of the composition of obtained liposomes. It has been revealed, that modification of DPPC liposomes by these amphiphiles results in increase of the stability of formulation, which could be extended from 2 weeks up to 5 months. Obtained liposomes have been used for encapsulation of hydrophilic cargos – antibiotic chloramphenicol and anticancer drug cisplatin and their in vitro release profiles have been evaluated. Liposomal formulation of chloramphenicol has been tested for hemolytic activity, which allowed to demonstrate its 4-fold lower toxicity in comparison with individual amphiphile of the same concentration. Examination of liposomal formulation of cisplatin against HeLa cancer cells has revealed, that encapsulated drug is more effective, than free one by about 5 times. Using confocal microscopy technique, it has been shown, that modification of liposomes with a hydroxyethylated imidazolium amphiphiles results in targeted delivery of encapsulated compounds to mitochondria.
Colocalization of cascade enzymes is broadly discussed as a phenomenon that can boost the cascade reaction throughput, although a direct experimental verification is often challenging. This is mainly ...due to difficulties in establishing proper size regimes and in the analytical quantification of colocalization effect with adequate experimental systems and simulations. In this study, by taking advantage of reversible DNA-directed colocalization of enzymes on microspheres, we established a cascade system that can be used to directly evaluate the colocalization effect with exactly the same experimental settings except for the state of enzyme dispersion. In the regime of highly dilute microspheres of particular sizes, the colocalized cascade shows enhanced activity compared with the freely diffusing cascade, as evidenced by a shortened lag phase in the time-course production. Reaction-diffusion modeling reveals that the enhancement can be ascribed to the initial accumulation of intermediate substrate around the colocalized enzymes and is found to be carrier-size-dependent. This work demonstrates the dependence of the colocalization effect of enzyme cascades on an interplay of nano- and microscales, lending theoretical support to the rational design of highly efficient multienzyme catalysts.
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