Optoelectronic Properties and Fluorescence Lifetime Imaging Application of Donor-Acceptor Dyads Derived From 2,6-DicarboxyBODIPY.

Donor-acceptor BODIPY dyads, functionalized at the 2 and 6 positions with benzyl ester (BDP-DE) or carboxylic acid (BDP-DA) groups, were synthesized, and their optoelectronic properties were investigated. Carbonyl groups were found to increase the reduction potential of the BODIPY core by 0.15-0.

Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients.

Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR O-sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts.

Fluorescence Intensity and Fluorescence Lifetime Imaging Microscopies (FLIM) of Cell Differentiation in the Small Intestinal Organoids Using Cholera Toxin.

Live cell microscopies of in vitro, ex vivo, and in vivo experimental intestinal models enable visualizing cell proliferation, differentiation, and functional cellular status in response to intrinsic and extrinsic (e.g., in the presence of microbiota) factors.

Balance between the cell viability and death in 3D.

Cell death is a phenomenon, frequently perceived as an absolute event for cell, tissue and the organ. However, the rising popularity and complexity of such 3D multicellular 'tissue building blocks' as heterocellular spheroids, organoids, and 'assembloids' prompts to revise the definition and quantification of cell viability and death. It raises several questions on the overall viability of all the cells within 3D volume and on choosing the appropriate, continuous, and non-destructive viability assay enabling for a single-cell analysis.

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids.

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented.

Extracellular Ca-Sensing Fluorescent Protein Biosensor Based on a Collagen-Binding Domain.

The importance of extracellular gradients of biomolecules is increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, the dynamics of extracellular calcium concentration is rarely studied. Here, we present a low affinity Ca biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides.

Visualization of Stem Cell Niche by Fluorescence Lifetime Imaging Microscopy.

Fluorescence lifetime imaging microscopy (FLIM), enabling live quantitative multiparametric analyses, is an emerging bioimaging approach in tissue engineering and regenerative medicine. When combined with stem cell-derived intestinal organoid models, FLIM allows for tracing stem cells and monitoring of their proliferation, metabolic fluxes, and oxygenation. It is compatible with the use of live Matrigel-grown intestinal organoids produced from primary adult stem cells, crypts, and transgenic Lgr5-GFP mice.

A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses.

Stem cells and the niche in which they reside feature a complex microenvironment with tightly regulated homeostasis, cell-cell interactions and dynamic regulation of metabolism. A significant number of organoid models has been described over the last decade, yet few methodologies can enable single cell level resolution analysis of the stem cell niche metabolic demands, in real-time and without perturbing integrity. Here, we studied the redox metabolism of Lgr5-GFP intestinal organoids by two emerging microscopy approaches based on luminescence lifetime measurement - fluorescence-based FLIM for NAD(P)H, and phosphorescence-based PLIM for real-time oxygenation.

Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy.

Monitoring of cell metabolism represents an important application area for fluorescence lifetime imaging microscopy (FLIM). In particular, assessment of mitochondrial membrane potential (MMP) in complex three-dimensional multicellular in vitro, ex vivo, and in vivo models would enable improved segmentation and functional discrimination of cell types, directly report on the mitochondrial function and complement the quenched-phosphorescence detection of cellular O and two-photon excited FLIM of endogenous NAD(P)H. Here, we report the green and orange-emitting fluorescent dyes SYTO and tetramethylrhodamine methyl ester (TMRM) as potential FLIM probes for MMP.

Properties of a cryptic lysyl oxidase from haloarchaeon .

Background: Lysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic.

Methods: LOX open reading frame was cloned from in an expression vector. Recombinant lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding.

Nanoparticle-based fluoroionophore for analysis of potassium ion dynamics in 3D tissue models and .

The imaging of real-time fluxes of K ions in live cell with high dynamic range (5-150 mM) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney and other tissues. In particular, the research on high-performance deep-red fluorescent nanoparticle-based biosensors is highly anticipated. We found that BODIPY-based FI3 K-sensitive fluoroionophore encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids.

Cellulose-based scaffolds for fluorescence lifetime imaging-assisted tissue engineering.

Quantitative measurement of pH and metabolite gradients by microscopy is one of the challenges in the production of scaffold-grown organoids and multicellular aggregates. Herein, we used the cellulose-binding domain (CBD) of the Cellulomonas fimi CenA protein for designing biosensor scaffolds that allow measurement of pH and Ca gradients by fluorescence intensity and lifetime imaging (FLIM) detection modes. By fusing CBD with pH-sensitive enhanced cyan fluorescent protein (CBD-ECFP), we achieved efficient labeling of cellulose-based scaffolds based on nanofibrillar, bacterial cellulose, and decellularized plant materials.

Multi-Parametric Imaging of Hypoxia and Cell Cycle in Intestinal Organoid Culture.

Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O distribution in mouse intestinal organoids.

Live cell imaging of mouse intestinal organoids reveals heterogeneity in their oxygenation.

Intestinal organoids are widely applied in stem cell research, regenerative medicine, toxicology, pharmacology, and host-microbe interactions research. The variability of oxidative metabolism for stem and differentiated cell types constituting organoid is known to be important but so far it has not been studied in details. Here, we report the use of live cell microscopy of oxygen via the phosphorescence lifetime imaging microscopy (PLIM) method to address the oxygenation and variability of aerobic metabolism of individual organoids in the culture.

Phosphorescent Oxygen and Mechanosensitive Nanostructured Materials Based on Hard Elastic Polypropylene Films.

It is well known that sensitivity of quenched-phosphorescence O sensors can be tuned by changing the nature of indicator dye and host polymer acting as encapsulation and quenching mediums. Here, we describe a new type of sensor materials based on nanostructured hard elastic polymeric substrates. With the example of hard elastic polypropylene films impregnated with Pt-benzoporphyrin dye, we show that such substrates enable simple one-step fabrication of O sensors by standard and scalable polymer processing technologies.

Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase.

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.

Quantitative analysis of mucosal oxygenation using ex vivo imaging of healthy and inflamed mammalian colon tissue.

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O in freshly prepared samples of healthy and inflamed colon tissue.

Evolutionary diversification of the BetaM interactome acquired through co-option of the ATP1B4 gene in placental mammals.

ATP1B4 genes represent a rare instance of orthologous vertebrate gene co-option that radically changed properties of the encoded BetaM proteins, which function as Na,K-ATPase subunits in lower vertebrates and birds. Eutherian BetaM has lost its ancestral function and became a muscle-specific resident of the inner nuclear membrane. Our earlier work implicated BetaM in regulation of gene expression through direct interaction with the transcriptional co-regulator SKIP.

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-μm) umbrella cells located superficially in the urinary bladder epithelium.

A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression.

Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways.

[P4-ATP-ase Atp8b1/FIC1: structural properties and (patho)physiological functions].

P4-ATP-ases comprise an interesting family among P-type ATP-ases, since they are thought to play a major role in the transfer of phospholipids such as phosphatydylserine from the outer leaflet to the inner leaflet. Isoforms of P4-ATP-ases are partially interchangeable but peculiarities of tissue-specific expression of their genes, intracellular localization of proteins, as well as regulatory pathways lead to the fact that, on the organismal level, serious pathologies may develop in the presence of structural abnormalities in certain isoforms. Among P4-ATP-ases a special place is occupied by ATP8B1, for which several mutations are known that lead to serious hereditary diseases: two forms of congenital cholestasis (PFIC1 or Byler disease and benign recurrent intrahepatic cholestasis) with extraliver symptoms such as sensorineural hearing loss.

Nuclear translocation of lysyl oxidase is promoted by interaction with transcription repressor p66β.

Lysyl oxidase (LOX) is an amine oxidase involved in protein cross-linking of the extracellular matrix. Less well characterized is the role that LOX plays among nuclear proteins, and molecular mechanisms of its transport to the nucleus are currently unknown. Here, we have employed yeast two-hybrid library screening and found that the LOX catalytic domain interacts with the transcription repressor p66β.

Two distinct nuclear localization signals in mammalian MSL1 regulate its function.

MSL1 protein regulates global histone H4 acetylation at residue K16 in stem and cancer cells, through interaction with KAT8. The functional significance of mammalian MSL1 isoforms, involved in various protein interactions, is poorly understood. We report the identification of a novel nuclear localization signal (NLS), common to all MSL1 isoforms, in addition to previously known bipartite NLS, located in domain PEHE.

Control of lysyl oxidase activity through site-specific deuteration of lysine.

Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE).