Integrated Metabolomics Approach Reveals the Dynamic Variations of Metabolites and Bioactivities in Paeonia ostii 'Feng Dan' Leaves during Development.

Paeonia ostii 'Feng Dan' is widely cultivated in China for its ornamental, medicinal, and edible properties. The whole plant of tree peony is rich in bioactive substances, while the comprehensive understanding of metabolites in the leaves is limited. In this study, an untargeted metabolomics strategy based on UPLC-ESI-TOF-MS was conducted to analyze the dynamic variations of bioactive metabolites in P. ostii 'Feng Dan' leaves during development. A total of 321 metabolites were rapidly annotated based on the GNPS platform, in-house database, and publications. To accurately quantify the selected metabolites, a targeted method of HPLC-ESI-QQQ-MS was used. Albiflorin, paeoniflorin, pentagalloylglucose, luteolin 7-glucoside, and benzoylpaeoniflorin were recognized as the dominant bioactive compounds with significant content variations during leaf development. Metabolite variations during the development of P. ostii 'Feng Dan' leaves are greatly attributed to the variations in antioxidant activities. Among all tested bacteria, the leaf extract exhibited exceptional inhibitory effects against Streptococcus hemolytis-ß. This research firstly provides new insights into tree peony leaves during development. The stages of S1-S2 may be the most promising harvesting time for potential use in food or pharmaceutical purposes.

Evaluating Natural Language Processing Packages for Predicting Hospital-Acquired Pressure Injuries From Clinical Notes.

Incidence of hospital-acquired pressure injury, a key indicator of nursing quality, is directly proportional to adverse outcomes, increased hospital stays, and economic burdens on patients, caregivers, and society. Thus, predicting hospital-acquired pressure injury is important. Prediction models use structured data more often than unstructured notes, although the latter often contain useful patient information. We hypothesize that unstructured notes, such as nursing notes, can predict hospital-acquired pressure injury. We evaluate the impact of using various natural language processing packages to identify salient patient information from unstructured text. We use named entity recognition to identify keywords, which comprise the feature space of our classifier for hospital-acquired pressure injury prediction. We compare scispaCy and Stanza, two different named entity recognition models, using unstructured notes in Medical Information Mart for Intensive Care III, a publicly available ICU data set. To assess the impact of vocabulary size reduction, we compare the use of all clinical notes with only nursing notes. Our results suggest that named entity recognition extraction using nursing notes can yield accurate models. Moreover, the extracted keywords play a significant role in the prediction of hospital-acquired pressure injury.

Investigate the multipath erasure of nitrobenzene over nanoscale zero-valent-iron/N-doped biochar hybrid with extraordinary reduction performance.

In this study, the facile carbothermal reduction method was enforced using urea as dopant to modify the structure and chemical composition of nanoscale zero-valent-iron/biochar hybrid thereby boosting its reduction performance. Through fine-tuning the N-doped amount, the optimal nZVI/N-doped BC was obtained, which exhibited more active sites (nZVI, persistent free radicals (PFRs), pyrrolic-N) and superior electrochemical conductivity. With these blessings, the electrons originating from galvanic cell reaction could zip along the highway within the hybrid. Taking nitrobenzene (NB) as the target pollutant, the quantitative analysis revealed that the NB reduction and adsorption removal efficiency were dramatically improved by 2.42 and 2.78 times, respectively. What's more, combining the in-situ experimental detection and theoretical calculations, unexpected NB reductive multipath with respect to PFRs and pyrrolic-N accelerating the Fe3+/Fe2+ cycle within the nZVI/N-doped BC system was decoded. The enhancement of Fe3+/Fe2+ cycle improved the electron utilization efficiency and maintained the reduction reactivity of the hybrid. This work raised awareness of the mechanisms regarding the reduction performance of nZVI/N-doped BC elevated by N-doped and the pollutant reductive pathway within the system, uncovered the dusty roles of PFRs and N-species during the reduction process.

Accuracy of newer generation intraocular lens power calculation formulas in pediatric cataract patients.

PURPOSE: To evaluate the accuracy of newer generation intraocular lens (IOL) power calculation formulas (EVO 2.0 and Kane) with established formulas (Barrett Universal II, Haigis and SRK/T) in pediatric cataract patients. METHODS: Retrospective study. We enrolled 110 eyes (110 patients) in Eye Hospital of Wenzhou Medical University. All patients underwent uneventful cataract surgery and implanted with posterior chamber IOL in the bag. We calculate the mean prediction errors (PE) and percentage within 1 diopter (D) at 1 month to assess the accuracy, and percentage > 2D was defined as prediction accident. Then, we performed subgroup analysis according to age and axial length (AL). RESULTS: The mean age and AL were 37.45 ± 23.28 months and 21.16 ± 1.29 mm. The mean PE for all patients was as follows: Barrett (- 0.30), EVO (0.18), Haigis (- 0.74), Kane (- 0.36), and SRK/T (0.58), p < 0.001. In addition, EVO and SRK/T formulas were relatively accurate in patients younger than 24 months and with AL ≤ 21 mm, while EVO got lower prediction accident rate than SRK/T (3/41 vs 8/41, 4/52 vs 5/52). Moreover, Barrett, EVO, and Kane formulas achieved better accuracy and lower prediction accident rate in patients older than 24 months and with AL > 21 mm (both > 51/69 and 43/58, and < 3/69 and 3/58). CONCLUSIONS: In patients older than 24 months and with AL > 21 mm, Barrett, EVO, and Kane formulas were relatively accurate, while in patients younger than 24 months and with AL ≤ 21 mm, EVO was more accurate, followed by SRK/T formula.

Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset.

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

Recognition-Enabled Automated Analyte Identification via 19F NMR.

Nuclear magnetic resonance (NMR) is an indispensable tool for structural elucidation and noninvasive analysis. Automated identification of analytes with NMR is highly pursued in metabolism research and disease diagnosis; however, this process is often complicated by the signal overlap and the sample matrix. We herein report a detection scheme based on 19F NMR spectroscopy and dynamic recognition, which effectively simplifies the detection signal and mitigates the influence of the matrix on the detection. It is demonstrated that this approach can not only detect and differentiate capsaicin and dihydrocapsaicin in complex real-world samples but also quantify the ibuprofen content in sustained-release capsules. Based on the 19F signals obtained in the detection using a set of three 19F probes, automated analyte identification is achieved, effectively reducing the odds of misrecognition caused by structural similarity.

Toward Nanomolar Multi-Component Analysis by 19F NMR.

The widespread application of nuclear magnetic resonance (NMR) spectroscopy in detection is currently hampered by its inherently low sensitivity and complications resulting from the undesired signal overlap. Here, we report a detection scheme to address these challenges, where analytes are recognized by 19F-labeled probes to induce characteristic shifts of 19F resonances that can be used as "chromatographic" signatures to pin down each low-concentration analyte in complex mixtures. This unique signal transduction mechanism allows detection sensitivity to be enhanced by using massive chemically equivalent 19F atoms, which was achieved through the proper installation of nonafluoro-tert-butoxy groups on probes of high structural symmetry. It is revealed that the binding of an analyte to the probe can be sensed by as many as 72 chemically equivalent 19F atoms, allowing the quantification of analytes at nanomolar concentrations to be routinely performed by NMR. Applications on the detection of trace amounts of prohibited drug molecules and water contaminants were demonstrated. The high sensitivity and robust resolving ability of this approach represent a first step toward extending the application of NMR to scenarios that are now governed by chromatographic and mass spectrometry techniques. The detection scheme also makes possible the highly sensitive non-invasive multi-component analysis that is difficult to achieve by other analytical methods.

Directing on-surface polymerization via a substrate-directed molecular template.

Using a template to control the on-surface polymerization process is valuable for building functional molecular nanostructures. Here, the role of the symmetric matching between a halogen-ligand component (H2TBrPP) and the substrate for the fabrication of a regular metal-organic structure on Cu(111) and Cu(100) surfaces was studied using scanning tunnelling microscopy (STM). Considering the formation of short-range order polymers on the Au(111) surface via the process of debromination due to the weak directing effect from the substrate to the precursors, a bilayer of ordered assembled structure of H2TBrPP/Au(111) has been fabricated and the molecules in the top layer are guided by the first-layer molecules. Owing to the steering effect of the substrate-directed molecular template, the H2TBrPP components in the top layer were polymerized into ordered molecular chain arrays along the given direction that is determined by the initial close-packed assembled structure of H2TBrPP components during the post-annealing treatment.

Optimal fermentation time for Nigrospora-fermented tea rich in bostrycin.

BACKGROUND: Bostrycin has many biological functions, such as anticancer activity, and is becoming increasingly popular. Nigrospora sphaerica HCH285, which has the ability to produce high levels of bostrycin, can be used to ferment sun-dried green tea of Camellia sinensis through acclimation, resulting in the development of a Nigrospora-fermented tea. The effects of fermentation time on the production of bostrycin by the HCH285 strain were investigated. RESULTS: After 45 days of fermentation, the bostrycin content reached 3.18 g kg-1 , which is the highest level during the whole fermentation. At 50 days, the tea liquor was red, had a strong mushroom odour and a sweet taste, and presented optimal quality. The contents of free amino acids, tea polyphenols and soluble sugars in the fermented tea decreased generally during the fermentation, although the content of water-soluble substances increased. Additionally, the results of a 14-day acute oral toxicity test showed that Nigrospora-fermented tea was nontoxic. CONCLUSION: The optimum fermentation time of Nigrospora-fermented tea was concluded to be 45-50 days. These results provide insights with respect to the development of tea biotechnology and new tea products with active ingredients. © 2020 Society of Chemical Industry.

Butyrophilin3A proteins and Vγ9Vδ2 T cell activation.

Despite playing critical roles in the immune response and having significant potential in immunotherapy, γδ T cells have garnered little of the limelight. One major reason for this paradox is that their antigen recognition mechanisms are largely unknown, limiting our understanding of their biology and our potential to modulate their activity. One of the best-studied γδ subsets is the human Vγ9Vδ2T cell population, which predominates in peripheral blood and can combat both microbial infections and cancers. Although it has been known for decades that Vγ9Vδ2T cells respond to the presence of small pyrophosphate-based metabolites, collectively named phosphoantigens (pAgs), derived from microbial sources or malignant cells, the molecular basis for this response has been unclear. A major breakthrough in this area came with the identification of the Butyrophilin 3A (BTN3A) proteins, members of the Butyrophilin/Butyrophilin-like protein family, as mediators between pAgs and Vγ9Vδ2T cells. In this article, we review the most recent studies regarding pAg activation of human Vγ9Vδ2T cells, mainly focusing on the role of BTN3A as the pAg sensing molecule, as well as its potential impact on downstream events of the activation process.

Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on Vγ9Vδ2 T cell activation.

Human Vγ9Vδ2 T cells respond to microbial infections as well as certain types of tumors. The key initiators of Vγ9Vδ2 activation are small, pyrophosphate-containing molecules called phosphoantigens (pAgs) that are present in infected cells or accumulate intracellularly in certain tumor cells. Recent studies demonstrate that initiation of the Vγ9Vδ2 T cell response begins with sensing of pAg via the intracellular domain of the butyrophilin 3A1 (BTN3A1) molecule. However, it is unknown how downstream events can ultimately lead to T cell activation. Here, using NMR spectrometry and molecular dynamics (MD) simulations, we characterize a global conformational change in the B30.2 intracellular domain of BTN3A1 induced by pAg binding. We also reveal by crystallography two distinct dimer interfaces in the BTN3A1 full-length intracellular domain, which are stable in MD simulations. These interfaces lie in close proximity to the pAg-binding pocket and contain clusters of residues that experience major changes of chemical environment upon pAg binding. This suggests that pAg binding disrupts a preexisting conformation of the BTN3A1 intracellular domain. Using a combination of biochemical, structural, and cellular approaches we demonstrate that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminishes pAg-induced T cell activation. Based on these results, we propose a model in which a conformational change in BTN3A1 is a key event of pAg sensing that ultimately leads to T cell activation.

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

A Photo-Crosslinkable Biotin Derivative of the Phosphoantigen (E)-4-Hydroxy-3-Methylbut-2-Enyl Diphosphate (HMBPP) Activates Vγ9Vδ2 T Cells and Binds to the HMBPP Site of BTN3A1.

Vγ9Vδ2 T cells play an important role in the cross talk of the innate and adaptive immune system. For their activation by phosphoantigens (PAgs), both cell surface receptors, the eponymous Vγ9Vδ2 T cell antigen receptors (Vγ9Vδ2 TCRs) on Vγ9Vδ2 T cells and butyrophilin 3A1 (BTN3A1) on the phosphoantigen-"presenting" cell, are mandatory. To find yet undetected but further contributing proteins, a biotinylated, photo-crosslinkable benzophenone probe BioBP-HMBPP (2) was synthesized from a known allyl alcohol in nine steps and overall 16 % yield. 2 is based on the picomolar PAg (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1). Laser irradiation of 2 at 308 nm initiated the photo-crosslinking reaction with proteins. When the B30.2 domain of BTN3A1, which contains a positively charged PAg-binding pocket, was exposed to increasing amounts of HMBPP (1), labeling by BioBP-HMBPP (2) was reduced significantly. Because BSA labeling was not impaired, 2 clearly binds to the same site as natural ligand 1. Thus, BioBP-HMBPP (2) is a suitable tool to identify co-ligands or receptors involved in PAg-mediated T cell activation.

Human gamma delta T cells: Evolution and ligand recognition.

The γδ T cell lineage in humans remains much of an enigma due to the low number of defined antigens, the non-canonical ways in which these cells respond to their environment and difficulty in tracking this population in vivo. In this review, we survey a comparative evolutionary analysis of the primate V, D and J gene segments and contrast these findings with recent progress in defining antigen recognition by different populations of γδ T cells in humans. Signatures of both purifying and diversifying selection at the Vδ and Vγ gene loci are placed into context of Vδ1+ γδ T cell recognition of CD1d presenting different lipids, and Vγ 9Vδ2 T cell modulation by pyrophosphate-based phosphoantigens through the butyrophilins BTN3A. From this comparison, it is clear that co-evolution between γδ TCRs and these ligands is likely occurring, but the diversity inherent in these recombined receptors is an important feature in ligand surveillance.

Voxel model of individual cells and its implementation in microdosimetric calculations using GEANT4.

Accurate dosimetric calculations at cellular and sub-cellular levels are crucial to obtain an increased understanding of the interactions of ionizing radiation with a cell and its nucleus and cytoplasm. Ion microbeams provide a superior opportunity to irradiate small biological samples, e.g., DNA, cells, and to compare their response to computer simulations. However, the phantoms used to simulate small biological samples at cellular levels are often simplified as simple volumes filled with water. As a first step to improve the situation in comparing measurements of cell response to ionizing radiation with model calculations, a realistic voxel model of a KB cell was constructed and used together with an already constructed geometry and tracking 4 (GEANT4) model of the horizontal microbeam line of the Centre d'Etudes Nucléaires de Bordeaux-Gradignan (CENBG) 3.5 MV Van de Graaf accelerator at the CENBG, France. The microbeam model was then implemented into GEANT4 for simulations of the average number of particles hitting an irradiated cell when a specified number of particles are produced in the beam line. The result shows that when irradiating the developed voxel model of a KB cell with 200 α particles, with a nominal energy of 3 MeV in the beam line and 2.34 MeV at the cell entrance, 100 particles hit the cell on average. The mean specific energy is 0.209 ± 0.019 Gy in the nucleus and 0.044 ± 0.001 Gy in the cytoplasm. These results are in agreement with previously published data, which indicates that this model could act as a reference model for dosimetric calculations of radiobiological experiments, and that the proposed method could be applied to build a cell model database.

From waste to catalyst: Growth mechanisms of ZSM-5 zeolite from coal fly ash & rice husk ash and its performance as catalyst for tetracycline degradation in fenton-like oxidation.

Coal fly ash (CFA), an industrial solid waste, can be utilized to synthesize Zeolite Socony Mobil-5 (ZSM-5) by incorporating an external silica source. In this study, a series of ZSM-5 zeolites were synthesized using rice husk ash (RHA) as the primary silica source and CFA as the primary aluminum source under controlled hydrothermal reaction conditions, and the growth mechanism of ZSM-5 was investigated. The process of ZSM-5 growth was featured by the transformation of hyperpoly silico-aluminate in CFA and RHA into monomers. These monomers formed crystal nuclei connected in a five-membered ring structure under the influence of Tetrapropyl ammonium hydroxide (TPAOH). The surplus monomeric silica-aluminate grew on the nucleus surface due to the addition of the silica source within RHA (RHA-SiO2), ultimately resulting in the development of ZSM-5 zeolite. Characterization results demonstrated that RHA-SiO2 exhibited favorable physical and chemical properties during the ZSM-5 synthesis, with a crystallinity of 99.03%, a specific surface area of 321.19 m2/g, a weight loss of only 3.06% at 800 °C and a total acidity of 0.65 mmol/g. To evaluate the catalytic performance of ZSM-5, Fe/Cu-modified ZSM-5 was developed and used as the catalyst for the degradation of tetracycline (TC) in Fenton-like oxidation. The results indicated that Fe/Cu-ZSM-5 exhibited excellent activity and stability as the catalyst for TC degradation and mineralization. The maximum TC degradation rate reached 99.02% in 10 min and the TOC removal could be up to 69.32% in 2 h. Characterization results indicated that the Fe/Cu ions redox cycle accelerated the generation of active species (1O2 and ˙OH) in Fenton-like systems. The ZSM-5 zeolite synthesized from solid waste demonstrated superb stability and catalytic activity, leading to the effective removal of TC. Since real wastewater generally contains various pollutants, future research efforts should focused on multi-pollutant treatment.

Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5.

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

On-bead purification and nanodisc reconstitution of human chemokine receptor complexes for structural and biophysical studies.

Chemokine receptors, a subfamily of G-protein-coupled receptors (GPCRs), are responsible for cell migration during physiological processes as well as in diseases like inflammation and cancers. Here, we present a protocol for solubilizing, purifying, and reconstituting complexes of chemokine receptors with their ligands in "nanodiscs," soluble lipid bilayers that mimic the native environment of membrane receptors. The protocol yields chemokine receptor complexes with sufficient purity and yield for structural and biophysical studies and should be applicable to other GPCRs.

Congenital cataracts affect the retinal visual cycle and mitochondrial function: A multi-omics study of GJA8 knockout rabbits.

Congenital cataracts are a threat to visual development in children, and the visual impairment persists after surgical treatment; however, the mechanisms involved remain unclear. Previous clinical studies have identified the effect of congenital cataracts on retinal morphology and function. To further understand the molecular mechanisms by which congenital cataracts affect retinal development, we analyzed retina samples from 7-week-old GJA8-knockout rabbits with congenital cataracts and controls by four-dimensional label-free quantification proteomics and untargeted metabolomics. Bioinformatics analysis of proteomic data showed that retinol metabolism, oxidative phosphorylation, and fatty acid degradation pathways were downregulated in the retinas of rabbits with congenital cataracts, indicating that their visual cycle and mitochondrial function were affected. Additional validation of differentially abundant proteins related to the visual cycle and mitochondrial function was performed using Parallel reaction monitoring and western blot experiments. Untargeted metabolome analysis showed significant upregulation of the antioxidant glutathione and ascorbic acid in the retinas of rabbits with congenital cataracts, indicating that their oxidative stress balance was not dysregulated. SIGNIFICANCE: Congenital cataracts in children can alter retinal structure and function, yet the mechanisms are uncertain. Here is the first study to use proteomics and metabolomics approaches to investigate the effects of congenital cataracts on retinal development in the early postnatal period. Our findings suggest that congenital cataracts have an impact on the retinal visual cycle and mitochondrial function. These findings give insight on the molecular pathways behind congenital cataract-induced visual function impairment in the early postnatal period.

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics.

CC chemokine receptor 5 (CCR5) contributes to inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A drug against CCR5 has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of inflammatory diseases and cancer has had limited success because of the complex biology and pharmacology of this receptor. CCR5 is activated by many natural and engineered chemokines that elicit distinct receptor signaling and trafficking responses, including some that sequester the receptor inside the cell. The sequestration phenomenon may be therapeutically exploitable, but the mechanisms by which different ligands traffic CCR5 to different cellular locations are poorly understood. Here we employed live cell ascorbic acid peroxidase proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveals distinct ligand-dependent CCR5 trafficking patterns with temporal resolution. All three chemokines internalize CCR5 via ß-arrestin- dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and with varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the molecular mechanisms behind CCR5 intracellular sequestration and suggest actionable patterns for the development of chemokine-based CCR5 targeting molecules. Significance Statement: CCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and HIV transmission. Along with its functional diversity, different CCR5 ligands can induce distinct receptor signaling responses and trafficking behaviors; the latter includes intracellular receptor sequestration which offers a potential therapeutic strategy for inhibiting CCR5 function. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the molecular basis for receptor sequestration including information that can be exploited for the development of CCR5 targeting molecules that promote retention of the receptor inside the cell.