Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high‐throughput screening variant of the Mammalian Membrane Two‐Hybrid (MaMTH‐HTS) to map the protein–protein interactions of wild‐type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient‐specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient‐derived intestinal organoids has a significant effect on CFTR functional expression.

From its inception, cell culture has been a great scientific tool for researchers in many diverse fields. The advancement from monolayer 2D cultures into three-dimensional cellular systems enabled a better experimental tool, as the 3D culture mimics in vivo environments more closely. Cells are aggregated in clusters, allowing for more cell-to-cell interactions, cell migration, and differences in nutrient and oxygen availability. Spheroids and organoids are most commonly used and have proven themselves as models for a large number of analytical purposes. The simplicity of spheroid production is often a good starting point. Because organoids are more complex, they can provide better and more complete data, but they can be difficult to grow and maintain. With increasing concern about the applicability of data obtained from animal studies and questions regarding animal welfare, these can replace a large proportion of these models and provide accurate and rapid results. In this overview, aimed at someone looking for an introductory summary of the requirements and possibilities of different 3D culture approaches, we give the basic information on various uses of spheroids and organoids in different fields of science. Assays based on spheroids and organoids can be adapted for a range of applications, and their use will continue to grow.

A synthesis of two series of 3-substituted quinazolinones was performed utilizing a green chemistry approach, deep eutectic solvents and microwaves, namely. 2-Methyl-3-substituted-quinazolin-4(3H)-one derivatives were synthesized in a two-step reaction, using choline chloride:urea deep eutectic solvent (DES). 3-Substituted-quinazolin-4(3H)-ones were synthesized in one-pot one-step reaction of anthranilic acid, amines and orthoester in a microwave reactor. For the synthesis of 2-methyl-3-substituted-quinazolin-4(3H)-ones, first conventional synthesis of benzoxazinone, as an intermediate, was performed. Further, benzoxazinone in reaction with corresponding amines, in choline choline:urea deep eutectic solvent, furnished desired compounds. These procedures are based on green principles with the aim of developing synthetic routes for the potential antitumor agents. All compounds were characterized by LC/MS, ¹H NMR and ¹³C NMR spectral techniques. Compound 1 bearing trifluoromethoxyphenyl group showed promising activity against HuT-78 cell line with IC₅₀ of 51.4 ± 5.1 µM. GRAPHICAL ABSTRACT

The synthesis of novel 6-chloro/morpholino/amino/-9-sulfonylpurine derivatives was accomplished in two ways, either (i) involving the condensation reaction of 6-chloropurine with commercially available arylsulfonyl chlorides in acetone and the presence of aqueous KOH at 0 °C, followed by the substitution of C6-chlorine with morpholine, or (ii) employing a reversed synthetic approach where 6-morpholinopurine and commercially available adenine bases were reacted with the corresponding alkyl, 2-arylethene and arylsulfonyl chlorides giving the N9 sulfonylated products, the latter particularly used where prior nonselective sulfonylation was observed. In both approaches, the sulfonylation reaction occurred regioselectively at the purine N9 position lacking any concurrent N7 derivatives, except in the case of a smaller methyl substituent on SO₂ and the free amino group at C6 of the purine ring. The tautomeric features of initial N9 unsubstituted purines, as well as stability trends among the prepared N-9-sulfonylpurine derivates, were investigated using DFT calculations with an important conclusion that electron-donating C6 substituents are beneficial for the synthesis as they both promote the predominance of the desired N9 tautomers and help to assure the stability of the final products. The newly synthesized 6-morpholino and 6-amino-9-sulfonylpurine derivatives showed antiproliferative activity on human carcinoma, lymphoma, and leukemia cells. Among the tested compounds, 6-morpholino 17 and 6-amino 22 derivatives, with trans-β-styrenesulfonyl group attached at the N9 position of purine, proved to be the most effective antiproliferative agents, causing accumulation of leukemia cells in subG0 cell cycle phase. Graphical Abstract

Cell culture models are of central importance for the investigation of cellular metabolism, proliferation and stress responses. In this study, the effects of different concentrations of glucose (1 g/L vs. 4.5 g/L) and fetal bovine serum (FBS; 5%, 10%, 15%) on viability, mitochondrial function and autophagy are investigated in four human cell lines: MRC-5, HeLa, Caco-2 and SW-620. Cells were cultured in defined media for 72 h, and viability was assessed by LDH release, mitochondrial membrane potential using Rhodamine 123, ATP content by luminescence and autophagy activity by dual fluorescence staining. The results showed that HeLa and SW-620 cancer cells exhibited increased proliferation and mitochondrial activity under high glucose conditions, while low glucose media resulted in decreased ATP content and increased membrane permeability in HeLa cells. MRC-5 fibroblasts and Caco-2 cells showed greater resilience to nutrient stress, with minimal changes in LDH release and consistent proliferation. Autophagy was activated under all conditions, with a significant increase only in selected cell-medium combinations. These results highlight the importance of medium composition in influencing cellular bioenergetics and stress responses, which has implications for cancer research, metabolic disease modelling and the development of serum-free culture systems for regenerative medicine.