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The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N , N -dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation. The pancreatic extracellular matrix (ECM) is known to differ between species, age groups and physiological states, but its compositional changes throughout human life are not well understood. Here, the authors study how the proteome of pancreatic ECM changes during human development and maturation.

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo . This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.

Multiple p38 MAP kinase inhibitors have been developed for the treatment of inflammatory diseases such as rheumatoid arthritis, but their effectiveness has been limited due to toxicity and tachyphylaxis, leading to a lack of clinical benefit. Efforts have been made to circumvent this limitation by targeting individual substrates downstream of p38, including MK2 and MK5. This approach has failed to yield clinical benefit despite preclinical evidence of a therapeutic effect. We hypothesized that there is redundancy in the MAPK activating kinase family that would necessitate blocking multiple kinases to sufficiently impact inflammatory processes. We used heterobifunctional protein degraders that either specifically degraded MK2 selectively or degraded MK2/3/5 simultaneously to test the hypothesis, in addition to genetic approaches to enable knockdown. In human PBMCs, elimination of MK2/3/5 with heterobifunctional degraders resulted in full reduction of TLR4 or TLR7/8 induced TNFα, whereas MK2-specific degradation only attenuated TNFα biosynthesis. In contrast, both specific MK2 degradation and broad MK2/3/5 degradation inhibited TGF-β-induced collagen production in human fibroblasts. This observation was consistent with genetic deletions of MK2, MK3 and MK5 (singly and in combination) whereby single deletion of MK2, MK3 or MK5 attenuated lipopolysaccharide (LPS) induced TNFα production and had no effect on R848-induced TNFα production. Double deletion of MK2 and MK3 or MK2 and MK5 or MK2/3/5 triple deletion had a significantly greater effect on TNFα production regardless of stimulus. The combined data suggest cooperativity between MK2 and either MK3 or MK5 for efficient, cell context-dependent modulation of inflammatory responses.

Protein post-translational modifications (PTMs) involve the covalent chemical modifications of specific amino acid residues in proteins, which play crucial roles in protein physiochemical properties, structures and their physiological functions. Alterations in protein PTMs have been implicated in many serious diseases. Comprehensive study of disease-related protein PTMs is critical to explore their roles in the pathogenesis of diseases, contributing to the diagnosis and clinical treatment of diseases. Recently, given the capability of monitoring and identifying thousands of peptides simultaneously, mass spectrometry (MS) has evolved as a powerful tool in bottom-up proteomics, especially the protein PTM analysis.This dissertation is devoted to the development and application of novel electrospray ionization mass spectrometry (ESI-MS) based strategies for the in-depth profiling and quantitative analysis of several important protein PTMs as well as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) technique for the in-situ imaging of biomolecules. A portion of this dissertation describes the development of a novel biotin tag-assisted MS-based method, as the first method, for the in-depth profiling of citrullinated and homocitrullinated proteins in biological samples. Its utility is demonstrated through its application in mouse tissue-specific citrullination and homocitrullination analysis. We further demonstrate the combination of the biotin tag-assisted MS-based method with different quantitative techniques, enabling the simultaneously qualitative and quantitative analysis of citrullinated and homocitrullinated proteins from different biological samples for the first time. Meanwhile, quantitative proteomics and phosphoproteomics was performed to reveal the signaling pathways involved in the macrophages treated by lipopolysaccharides (LPS) and thapsigargin (TPG).This dissertation also highlights two projects focusing on in-situ imaging of biomolecules from tissue sections using MALDI MS imaging (MSI). MALDI MSI of mouse arteries undergoing restenosis revealed the involvement of many bioactive lipids in the progress of restenosis. In addition, a novel subatmospheric pressure ionization source has been coupled with MS for the high-resolution imaging of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. Application of this new SubAP/MALDI MS platform to FFPE mouse ovarian cancer tissue section unraveled the specific distribution of high-mannose N-glycans in the tumor region, suggesting potential association of this type of N-glycans with tumor progression.

To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFβ) and its signaling protein Smad3, known contributors to IH. TGFβ/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFβ/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies — a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.

Lower urinary tract symptoms (LUTS) are a range of irritative or obstructive symptoms that commonly afflict aging population. The diagnosis is mostly based on patient-reported symptoms and current medication often fails to completely eliminate these symptoms. There is a pressing need for objective non-invasive approaches to measure symptoms and understand disease mechanisms. We developed an in-depth workflow combining urine metabolomics analysis and machine learning bioinformatics to characterize metabolic alterations and support objective diagnosis of LUTS. Machine learning feature selection and statistical tests were combined to identify candidate biomarkers, which were statistically validated with leave-one-patient-out cross-validation and absolutely quantified by selected reaction monitoring assay. Receiver operating characteristic analysis showed highly-accurate prediction power of candidate biomarkers to stratify patients into disease or non-diseased categories. The key metabolites and pathways may be possibly correlated with smooth muscle tone changes, increased collagen content and inflammation, which have been identified as potential contributors to urinary dysfunction in humans and rodents. Periurethral tissue staining revealed a significant increase in collagen content and tissue stiffness in men with LUTS. Together, our study provides the first characterization and validation of LUTS urinary metabolites and pathways to support the future development of a urine-based diagnostic test for LUTS.

To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFβ) and its signaling protein Smad3, known contributors to IH. TGFβ/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFβ/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies -- a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.

The increasing global reliance on alternative water sources underscores the critical need for enhanced desalination efficacy. Covalent organic frameworks (COFs), with their ordered porosity and tunable architectures, hold immense potential for next-generation desalination membranes. However, current COF membranes often fail in efficient seawater desalination due to pore sizes largely exceeding hydrated monovalent ion dimensions. Here we present a structurally stable, ultramicroporous COF membrane for low-pressure reverse osmosis (RO) desalination, engineered through a hydrogen-bond fortification strategy. Rational introduction of phenolic hydroxyl adjacent to aldehyde moieties yielded β-ketoenamine configurations enriched with hydrogen bonds, promoting AB-stacking and enhanced crystallinity in Tp-Bth COF membranes. The resultant COF membranes achieved 99.6% sodium chloride rejection with 1.7 L m −2 h −1 bar −1 water permeability at 15 bar, demonstrating high-performance low-pressure RO desalination. Notably, these membranes exhibited high acid resistance, retaining their initial performance after 30 days in a solution at pH 3. This work demonstrates a hydrogen-bond-mediated strategy to precisely tailor COF pore architecture for high-performance desalination. Covalent organic frameworks are promising for desalinations; however, it is challenging to control pore size for efficient desalination. Here the authors design an ultra-microporous membrane by introducing hydrogen bonding networks that enhance the crystallinity and desalination performance.

Homomorphic signatures have important potential in cloud computing and data privacy protection, but there are still problems such as low signature efficiency, high overhead, and difficulty in instantiation. To solve these problems, an efficient leveled fully homomorphic signature scheme LFHSS with shortened signature values is constructed. The scheme is based on the GPV framework and the RSIS problem on the NTRU lattice. By using the Fast Fourier Sampling algorithm, it achieves efficient signatures with smaller size. It introduces a homomorphic trapdoor function and designs three basic evaluations: homomorphic addition, multiplication, and scalar multiplication. These operations enable the LFHSS scheme to support homomorphic evaluations of functions consisting of addition, multiplication, and scalar multiplication within a certain circuit depth. Additionally, it is proven to be strongly unforgeable, and the scheme is implemented in software with correctness testing and performance analysis conducted. The experimental results show that the security level of LFHSS is 1.14 times higher than that of BCFL23, and the signature generation speed is 300+ times faster, the homomorphic evaluation speed is 7.8 times faster, and the verification speed is 48K times faster than that of BCFL23. The signature length of LFHSS is only 4.86% of BCFL23. The work in this paper is of great significance to the design and application of homomorphic signatures.