Alexei Grichine

How the energy status of enteric progenitors controls neurogliogenesis and the subsequent formation of the complex enteric nervous system (ENS) remains poorly understood. We previously showed that the tumor suppressor kinase LKB1 is essential for postnatal ENS maintenance through amino acid homeostasis. Here, we investigated LKB1's functions during embryonic ENS formation using a genetically engineered mouse model with conditional Lkb1 inactivation in neural crest progenitors during gut colonization. Using advanced 3D imaging techniques on cleared tissue including light sheet microscopy and adaptive optics confocal microscopy, we found that Lkb1 loss impairs early neuronal differentiation followed by progressive glial degeneration, leading to hypoganglionosis and compromised digestive tissue integrity. Notably, Lkb1 inactivation induced a transient upregulation of the glial stress marker S100β during gestation, suggestive of a reactive glial state preceding glial loss. Consistent with this response, Lkb1 loss elevated oxidative stress in the digestive tract and in neural crest progenitors and their glial derivatives, triggering DNA damage and p53 activation. Although p53 ablation rescued glial specification in vitro and glial maintenance in vivo, it only partially restored ENS architecture in vivo without rescuing enteric neuron numbers. Together, these findings establish LKB1 as a critical metabolic checkpoint governing neuronal–glial balance during ENS development and suggest that dysregulated LKB1 signaling may contribute to human enteric neurogliopathies.

Two-photon luminescent bioprobes have emerged as powerful tools for in cellulo and in vivo imaging.

D ³ A versatile molecular chelator with a two-photon absorbing acetophenone moiety forms highly stable and inter complexes with Gd ³⁺ and Eu ³⁺ , that can be used for MRI or two-photon microscopy.

Eu³⁺-based conjugates with an Eu³⁺-sensitising ter-aryl amido–phenyl–picolinamide antenna exhibit superior two-photon absorption properties to those of the bi-aryl amido–phenyl–picolinamide antenna for improved microscopy of living cells.

Lanthanide(III) (Ln³⁺) complexes have desirable photophysical properties for optical bioimaging. However, despite their advantages over organic dyes, their use for microscopy imaging is limited by the high-energy UV excitation they require and their poor ability to cross the cell membrane and reach the cytosol. Here we describe a novel family of lanthanide-based luminescent probes, termed dTAT[Ln·L], based on (i) a DOTA-like chelator with a picolinate moiety, (ii) a two-photon absorbing antenna to shift the excitation to the near infrared and (ii) a dimeric TAT cell-penetrating peptide for cytosolic delivery. Several Tb³⁺ and Eu³⁺ probes were prepared and characterized. Two-photon microscopy of live cells was attempted using a commercial microscope with the three probes showing the highest quantum yields (>0.15). A diffuse Ln³⁺ emission was detected in most cells, which is characteristic of cytosolic delivery of the Ln³⁺ complex. The cytotoxicity of these three probes was evaluated and the IC₅₀ ranged from 7 μM to >50 μM. The addition of a single positive or negative charge to the antenna of the most cytotoxic compound was sufficient to lower significantly or suppress its toxicity under the conditions used for two-photon microscopy. Therefore, the design reported here provides excellent lanthanide-based probes for two-photon microscopy of living cells.

Cationic lanthanide complexes are generally able to spontaneously internalize into living cells. Following our previous works based on a diMe‐cyclen framework, a second generation of cationic water‐soluble lanthanide complexes based on a constrained cross‐bridged cyclam macrocycle functionalized with donor‐π‐conjugated picolinate antennas was prepared with europium(III) and ytterbium(III). Their spectroscopic properties were thoroughly investigated in various solvents and rationalized with the help of DFT calculations. A significant improvement was observed in the case of the Eu³⁺ complex, while the Yb³⁺ analogue conserved photophysical properties in aqueous solvent. Two‐photon (2P) microscopy imaging experiments on living T24 human cancer cells confirmed the spontaneous internalization of the probes and images with good signal‐to‐noise ratio were obtained in the classic NIR‐to‐visible configuration with the Eu³⁺ luminescent bioprobe and in the NIR‐to‐NIR with the Yb³⁺ one.

Although live imaging of dynamic processes in platelets is a challenging task, several important observations have been published during the last 20 years. We will discuss the amazing insights that have been achieved, the difficulties that can be encountered as well as some questions still open and the future technical perspectives.

Two trispicolinate 1,4,7‐triazacyclonane (TACN)‐based ligands bearing three picolinate biphotonic antennae were synthetized and their Yb³⁺ and Gd³⁺ complexes isolated. One series differs from the other by the absence (L¹)/presence (L²) of bromine atoms on the antenna backbone, offering respectively improved optical and singlet‐oxygen generation properties. Photophysical properties of the ligands, complexes and micellar Pluronic suspensions were investigated. Complexes exhibit high two‐photon absorption cross‐section combined either with NIR emission (Yb) or excellent ¹O₂ generation (Gd). The very large intersystem crossing efficiency induced by the combination of bromine atom and heavy rare‐earth element was corroborated with theoretical calculations. The ¹O₂ generation properties of L²Gd micellar suspension under two‐photon activation leads to tumour cell death, suggesting the potential of such structures for theranostic applications.

A family of europium (III) complexes based on a polydentate ligand functionalized by charge‐transfer antennae presents remarkable one‐ and two‐photon photophysical proper‐ties in water or buffer. A detailed analysis of their emission properties suggests that the wrapping of the ligand around the central rare‐earth ion results in an overall Cs symmetry in agreement with the theoretical simulation and that about 65–70 % of the emission intensity is concentrated in the hypersensitive ⁵D₀→⁷F₂ transition at 615 nm. Their brightness is excellent, in the range of the best lanthanide bioprobes making them very attractive for bio‐imaging experiments.