Shanghai Genomics, Inc.

A robust and versatile bioanalytical method is essential for the preclinical development of the novel caspase inhibitor F573 for acute liver failure.This endeavor faces significant challenges, including the need for high sensitivity to monitor rapid systemic clearance, the ability to handle diverse and complex biological matrices (e.g., plasma, tissues, excreta), and overcoming potential interferences from high plasma protein binding (>89 %). To address these challenges, a sensitive, selective, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of F573 across these matrices, with additional capability for qualitative metabolite profiling. The method used a simple protein precipitation protocol optimized for high-throughput processing. It was rigorously validated per FDA guidelines, demonstrating a low limit of quantification (LLOQ of 2 ng mL^-1 in 50 μL plasma), excellent precision (RSD <15 %), and accuracy. The method effectively addressed challenges from high plasma protein binding and matrix complexity. Its application enabled the first comprehensive characterization of F573's preclinical profile: it exhibited rapid absorption and elimination in rats and dogs, extensive tissue distribution, and limited blood-brain barrier penetration. Approximately 56 % of the administered dose was excreted unchanged, primarily via urinary and biliary routes. Furthermore, eight distinct metabolites were identified and profiled in rat samples. The developed LC-MS/MS method thus serves as a critical and reliable tool for the comprehensive preclinical pharmacokinetic evaluation of F573, underscoring its value in supporting the development of this promising therapeutic candidate.

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3^CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3^CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3^CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3^CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in the mature medaka testis. As for the interaction with transforming growth factor β-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired yeast 2-hybrid assay. Coimmunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastructural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (transforming growth factor β) and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA, which may be fundamentally conserved across the phyla during sexual differentiation.