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Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms. [Display omitted] • Trace the cellular origin of >5,900 proteins in locally enriched fluid compartments • Assess disease states at cellular resolution in non-regenerative organs in living humans • AI proteomic clocks reveal accelerated cell aging in non-age-related diseases • Molecular dysfunction of retinal cells during brain disease The integration of liquid-biopsy proteomics, cell-level transcriptomics, and artificial intelligence allows for non-destructive assessment of aging and disease at the cell level in non-regenerative organs, such as the eye, in living humans. [ABSTRACT FROM AUTHOR]

This protocol describes an ex vivo model to quantify enzymatic effects on vitreous liquefaction using porcine eyes in a transwell-plate system via induced syneresis. It provides a standardized dissection process and performs critical steps for gel-liquid separation with high precision, minimal tissue loss, and scalability. The protocol can be applied to other studies investigating vitreous liquefaction or gelatinous tissue analysis and can also serve to study vitreous liquefaction in vivo as it may occur during aging or disease progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Ocular drug implants (ODIs) are beneficial for treating ocular diseases. However, the lack of a robust injection approach for small-eyed model organisms has been a major technical limitation in developing ODIs. Here, we present a cost-effective, minimally invasive protocol to deliver ODIs into the mouse vitreous called Mouse Implant Intravitreal Injection (MI3). MI3 provides two alternative surgical approaches (air-pressure or plunger) to deliver micro-scaled ODIs into milli-scaled eyes, and expands the preclinical platforms to determine ODIs' efficacy, toxicity, and pharmacokinetics. For complete details on the use and execution of this protocol, please refer to Sun et al. (2021)., Competing Interests: The authors declare no competing interests.

Increased calpain activity is linked to neuroinflammation including a heritable retinal disease caused by hyper-activating mutations in the calcium-activated calpain-5 (CAPN5) protease. Although structures for classical calpains are known, the structure of CAPN5, a non-classical calpain, remains undetermined. Here we report the 2.8 Å crystal structure of the human CAPN5 protease core (CAPN5-PC). Compared to classical calpains, CAPN5-PC requires high calcium concentrations for maximal activity. Structure-based phylogenetic analysis and multiple sequence alignment reveal that CAPN5-PC contains three elongated flexible loops compared to its classical counterparts. The presence of a disease-causing mutation (c.799G>A, p.Gly267Ser) on the unique PC2L2 loop reveals a function in this region for regulating enzymatic activity. This mechanism could be transferred to distant calpains, using synthetic calpain hybrids, suggesting an evolutionary mechanism for fine-tuning calpain function by modifying flexible loops. Further, the open (inactive) conformation of CAPN5-PC provides structural insight into CAPN5-specific residues that can guide inhibitor design., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Conformational dynamics has an established role in enzyme catalysis, but its contribution to ligand binding and specificity is largely unexplored. Here we used the Tiam1 PDZ domain and an engineered variant (QM PDZ) with broadened specificity to investigate the role of structure and conformational dynamics in molecular recognition. Crystal structures of the QM PDZ domain both free and bound to ligands showed structural features central to binding (enthalpy), while nuclear-magnetic-resonance-based methyl relaxation experiments and isothermal titration calorimetry revealed that conformational entropy contributes to affinity. In addition to motions relevant to thermodynamics, slower microsecond to millisecond switching was prevalent in the QM PDZ ligand-binding site consistent with a role in ligand specificity. Our data indicate that conformational dynamics plays distinct and fundamental roles in tuning the affinity (conformational entropy) and specificity (excited-state conformations) of molecular interactions. More broadly, our results have important implications for the evolution, regulation, and design of protein-ligand interactions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)