Project description:Primary outcome(s): Comparison of diagnostic accuracy of urinary protein fragments and carcinoembryonic antigen (CEA) for adenocarcinoma (gastric cancer, colorectal cancer, or bile duct cancer)
Project description:Transparency in the lens is accomplished by the dense packing and short-range order interactions of the crystallin proteins in fiber cells lacking organelles. These features are accompanied by a lack of protein turnover, leaving lens proteins susceptible to a number of damaging modifications and aggregation. The loss of lens transparency is attributed in part to such aggregation during aging. Among the damaging post-translational modifications that accumulate in long-lived proteins, isomerization at aspartate residues has been shown to be extensive throughout the crystallins. In this study of the human lens, we localize the accumulation of l-isoaspartate within water-soluble protein extracts primarily to crystallin peptides in high-molecular weight aggregates and show with MS that these peptides are from a variety of crystallins. To investigate the consequences of aspartate isomerization, we investigated two αA crystallin peptides 52LFRTVLDSGISEVR65 and 89VQDDFVEIH98, identified within this study, with the l-isoaspartate modification introduced at Asp58 and Asp91, respectively. Importantly, whereas both peptides modestly increase protein precipitation, the native 52LFRTVLDSGISEVR65 peptide shows higher aggregation propensity. In contrast, the introduction of l-isoaspartate within a previously identified anti-chaperone peptide from water-insoluble aggregates, αA crystallin 66SDRDKFVIFL(isoAsp)VKHF80, results in enhanced amyloid formation in vitro The modification of this peptide also increases aggregation of the lens chaperone αB crystallin. These findings may represent multiple pathways within the lens wherein the isomerization of aspartate residues in crystallin peptides differentially results in peptides associating with water-soluble or water-insoluble aggregates. Here the eye lens serves as a model for the cleavage and modification of long-lived proteins within other aging tissues.
Project description:Lens epithelial cells (LECs) are crucial for lens transparency and function. Aging disrupts LEC homeostasis and contributes to the development of age-related cataracts (ARCs). The lens epithelium may rely on adult stem/progenitor cells to sustain homeostasis and counteract aging-related factors. While the existence of stem cells in the lens epithelium has been supported by various studies, identifying these cells remains challenging. Recent advancements in single-cell RNA sequencing (scRNA-seq) provide high-resolution insights into cellular heterogeneity and offer a promising approach to elucidate the presence, origin, and age-related changes of lens stem/progenitor cells. We collected human donor lenses with ethical approval for scRNA-seq analysis, including samples from four non-aged (<65 years) and four aged (>65 years) individuals. After quality assessment and filtering, the transcriptome profiles of a total of 56,717 cells from two groups were depicted and merged to allow identification of overlapping cell populations in the following analysis. Subsequent analyses involved cell (sub)type characterization, trajectory inference, and cell-cell communication. Six types of lens superficial cells were identified by scRNA-seq, including four categories of LECs: early differentiation state LECs (eLECs), middle differentiation state LECs (mLECs), late differentiation state LECs (lLECs), and transient amplifying cells (TACs), along with a small number of lens fibre cells and immune cells.
Project description:Mutations of the RNA-granule component TDRD7 (OMIM: 611258) cause pediatric cataract in humans. Here, we applied an integrated approach to elucidate the molecular pathology of cataract in Tdrd7 targeted-knockout (Tdrd7-/-) mice. Tdrd7-/- animals precipitously develop lens fiber cell abnormalities early in life, suggesting a global-level breakdown/mis-regulation of key cellular processes. High-throughput RNA-sequencing followed by iSyTE-integrated bioinformatics-based analysis identified the molecular chaperone and cytoskeletal-modulator, HSPB1 (HSP27), among the high-priority down-regulated candidates in Tdrd7-/- lens. Moreover, a protein 2-D fluorescence difference gel electrophoresis-coupled mass spectrometry screen also identified HSPB1 to be reduced in Tdrd7-/- lens, offering independent support for focusing efforts on this factor to explain Tdrd7-/- cataract. Reduction of HSPB1 preceded lens morphological abnormalities, suggesting that cytoskeletal defects underlie the Tdrd7-/- cataract phenotype. In agreement, scanning electron microscopy revealed abnormal fiber cell membrane protrusions in Tdrd7-/- lenses. Significantly, abnormal F-actin staining was detected specifically in Tdrd7-/- fiber cells that exhibit nuclear degradation, thereby revealing that there are distinct mechanisms based on pre- or post-nuclear degradation differentiation stage for F-actin cytoskeletal maintenance in fiber cells. Further, RNA-immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA-imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in a specific pre-nuclear degradation area of differentiating fiber cells, indicating that TDRD7-ribonucleoprotein complexes are necessary for controlling optimal levels of key factors in lens development. Together, these data uncover a novel role for TDRD7 in regulating elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts.
Project description:Foxp1 was strongly expressed in developing lens, and its knockout in lens resulted in failure of appropriate lens development. By microarray analysis, we examined effects of loss-of Foxp1 for gene expression pattern of lens at P0 developmental stage.