Comprehensive Glycomic and Glycoproteomic Analyses of Human Programmed Cell Death Protein 1 Extracellular Domain.

Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 N-glycosylation sites and 25 potential O-glycosylation sites, which implicates the importance of glycosylation. The structure of hPD-1 has been intensively studied, but the glycosylation of this protein, especially the glycan on each glycosylation site, has not been comprehensively illustrated. In this study, hPD-1 ECD expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells was analyzed; not only N- and O-glycosylation sites but also the glycans on these sites were comprehensively analyzed using mass spectrometry. In addition, hPD-1 ECD binding to different anti-hPD-1 antibodies was tested, and N-glycans were found functioned differently. All of this glycan information will be beneficial for future PD-1 studies.

Identification of a novel xanthine oxidoreductase inhibitor for hyperuricemia treatment with high efficacy and safety profile.

Hyperuricemia is with growing incidence and of high risk to develop into gout and other metabolic diseases. The key enzyme catalyzing uric acid synthesis, xanthine oxidoreductase (XOR) is a vital target for anti-hyperuricemic drugs, while XOR inhibitors characterized as both potent and safe are currently in urgent need. In this study, a novel small molecule compound, CC15009, was identified as a specific XOR inhibitor. CC15009 exerted strongest in vitro XOR inhibitory activity among current XOR inhibitors. It also showed favorable dose-dependent uric acid-lowering effects in two different XOR substrate-induced hyperuricemic mouse models, which was significantly superior than the current first-line drug, allopurinol. Mechanically, the direct binding of CC15009 against XOR was confirmed by molecular docking and SPR analysis. The inhibition mode was competitive and reversible. Besides, the potential antioxidant activity of CC15009 was indicated by its strong inhibitory activity against the oxidized isoform of XOR, which reduced ROS generation as the byproduct. Regarding the safety concerns of current XOR inhibitors, especially in cardiovascular risks, the safety of CC15009 was comprehensively evaluated. No significant abnormality was observed in the acute, subacute toxicity tests and mini-AMES test. Notably, there was no obvious inhibition of CC15009 against cardiac ion channels, including hERG, Nav1.5, Cav1.2 at the concentration of 30 µM, indicating its lower cardiovascular risk. Taken together, our results supported CC15009 as a candidate of high efficacy and safety profile to treat hyperuricemia through direct XOR inhibition.

Prior disulfide bond-mediated Ser/Thr ligation.

In this work, we developed a novel strategy, prior disulfide bond-mediated Ser/Thr ligation (PD-STL), for the chemical synthesis of peptides and proteins. This approach combines disulfide bond-forming chemistry with Ser/Thr ligation (STL), converting intermolecular STL into intramolecular STL to effectively proceed regardless of concentrations. We demonstrated the effectiveness of PD-STL under high dilution conditions, even for the relatively inert C-terminal proline at the ligation site. Additionally, we applied this method to synthesize the N-terminal cytoplasmic domain (2-104) of caveolin-1 and its Tyr14 phosphorylated form.

Applying a Racial Lens to the "Cycle of Violence".

One overlooked result in a 1989 Science paper on the "cycle of violence" was a race-specific increase in risk for arrest for violence among Black maltreated children, but not White maltreated children. We examine whether race differences in the cycle of violence are explained by risk factors traditionally associated with violence. Using a prospective design, maltreated and non-maltreated children were matched on age, sex, race, and approximate family social class and interviewed at mean age 28.7 years (N = 1196). Arrest histories were obtained through age 50.5. Regression analyses included maltreatment, race, self-reported violent behavior, and risk factors (e.g., family, school, neighborhood variables). For arrests for violent crime, race was a significant predictor, whereas childhood maltreatment was not significant. For violent arrests, there was a significant race × maltreatment interaction when the total number of risk factors were included controlling for self-reported violent behaviors. For self-reported violent behaviors, childhood maltreatment remained significant for some risk factors. However, race did not predict self-reported violent behaviors. Offending behavior and traditional risk factors did not explain the disproportionate arrests among Black maltreated children. This disparity in the cycle of violence may reflect complex processes influenced by racial bias or structural racism.

An integrated preventive and therapeutic magnetic nanoparticle loaded with rhamnolipid and vancomycin for combating subgingival biofilms.

OBJECTIVES: Mechanical debridement supplemented with antibacterial agents effectively eradicates subgingival biofilms formed in the periodontal pockets of severe periodontitis patients. However, the available antimicrobial agents have limited penetrating ability to kill the bacteria encased in the deep layers of biofilms. This study aimed to fabricate a novel magnetic nanoparticle (MNP) loaded with rhamnolipid (RL) and vancomycin (Vanc, Vanc/RL-Ag@Fe3O4) to combat subgingival biofilms. METHODS: The multispecies subgingival biofilm was formed by periodontal pathogens, including Streptococcus oralis (S. oralis), Streptococcus sanguinis (S. sanguinis), Actinomyces naeslundii (A. naeslundii), Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). Scanning electron microscope (SEM), confocal laser scanning microscopy (CLSM), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the anti-biofilm efficacy of Vanc/RL-Ag@Fe3O4 with or without a magnetic field on multispecies subgingival biofilms. RESULTS: The minimal inhibitory concentration (MIC) values of Vanc/RL-Ag@Fe3O4 on S. oralis, S. sanguinis, A. naeslundii, P. gingivalis, and F. nucleatum were 25, 50, 100, 50, and 25 µg/mL, respectively. Vanc/RL-Ag@Fe3O4 (200 µg/mL) reduced the 7-d biofilm thickness from 22 to 13 µm by degrading extracellular polymeric substance (EPS) and killing most bacteria except for tolerant F. nucleatum. A magnetic field enhanced the anti-biofilm effect of Vanc/RL-Ag@Fe3O4 by facilitating its penetration into the bottom layers of biofilms and killing tolerant F. nucleatum. SIGNIFICANCE: Vanc/RL-Ag@Fe3O4 MNPs can release RL, Vanc, and Ag and eradicate subgingival biofilms by disrupting EPS and killing bacteria. Vanc/RL-Ag@Fe3O4 combined with a magnetic force is a promising approach for combating periodontal infection.

Dissecting phospho-motif-dependent Shc1 interactome using long synthetic protein fragments.

Activated receptor tyrosine kinases (RTKs) rely on the assembly of signaling proteins into high-dimensional protein complexes for signal transduction. Shc1, a prototypical scaffold protein, plays a pivotal role in directing phosphotyrosine (pY)-dependent protein complex formation for numerous RTKs typically through its two pY-binding domains. The three conserved pY sites within its CH1 region (Shc1CH1) hold particular significance due to their substantial contribution to its functions. However, how Shc1 differentially utilizes these sites to precisely coordinate protein complex assembly remains unclear. Here, we employed multiple peptide ligation techniques to synthesize an array of long protein fragments (107 amino acids) covering a significant portion of the Shc1CH1 region with varying phosphorylation states at residues Y239, 240, 313, and S335. By combining these phospho-Shc1CH1 fragments with integrated proteomics sample preparation and quantitative proteomic analysis, we were able to comprehensively resolve the site-specific interactomes of Shc1 with single amino acid resolution. By applying this approach to different cancer cell lines, we demonstrated that these phospho-Shc1CH1 fragments can be effectively used as a diagnostic tool to assess cell type-specific RTK signaling networks. Collectively, these biochemical conclusions help to better understand the sophisticated organization of pY-dependent Shc1 adaptor protein complexes and their functional roles in cancer.

Clickable tryptophan modification for late-stage diversification of native peptides.

As the least abundant residue in proteins, tryptophan widely exists in peptide drugs and bioactive natural products and contributes to drug-target interactions in multiple ways. We report here a clickable tryptophan modification for late-stage diversification of native peptides, via catalyst-free C2-sulfenylation with 8-quinoline thiosulfonate reagents in trifluoroacetic acid (TFA). A wide range of groups including trifluoromethylthio (SCF3), difluoromethylthio (SCF2H), (ethoxycarbonyl)difluoromethylthio (SCF2CO2Et), alkylthio, and arylthio were readily incorporated. The rapid reaction kinetics of Trp modification and full tolerance with other 19 proteinogenic amino acids, as well as the super dissolving capability of TFA, render this method suitable for all kinds of Trp-containing peptides without limitations from sequences, hydrophobicity, and aggregation propensity. The late-stage modification of 15 therapeutic peptides (1.0 to 7.6 kilodaltons) and the improved bioactivity and serum stability of SCF3- and SCF2H-modified melittin analogs illustrated the effectiveness of this method and its potential in pharmacokinetic property improvement.

Trifluoroacetic Acid Mediated Additive-Free Late-Stage Native Peptide Cyclization to Form Disulfide Mimetics via Thioketalization with Ketones.

Peptide cyclization is often used to introduce conformational rigidity and to enhance the physiological stability of the peptide. This study presents a novel late-stage cyclization method for creating thioketal cyclic peptides from bis-cysteine peptides and drugs. Symmetrical cyclic ketones and acetone were found to react with bis-cysteine unprotected peptides efficiently to form thioketal linkages in trifluoroacetic acid (TFA) without any other additive. The attractive features of this method include high chemoselectivity, operational simplicity, and robustness. In addition, TFA as the reaction solvent can dissolve any unprotected peptide. As a showcase, the dimethyl thioketal versions of lanreotide and octreotide were prepared and evaluated, both of which showed much improved reductive stability and comparable activity.

Total Syntheses of Streptamidine and Klebsazolicin Using Biomimetic On-Resin Ring-Closing Amidine Formation.

Diketopiperazine (DKP) derived cyclic amidine structures widely exist in peptide natural products according to the genome mining result. The largely unknown bioactivity and mode of action are partially caused by the poor availability of the compounds via microbiological and chemical approaches. To tackle this challenge, in this work, we have developed the on-resin ring-closing amidine formation strategy to synthesize peptides containing N-terminal DKP derived cyclic amidine structure, in which the 6-exo-trig cyclization mediated by HgCl2 activation of thioamides was the key step. Leveraging from this new strategy, we finished the total syntheses of streptamidine and klebsazolicin. Meanwhile, eleven klebsazolicin analogues were synthesized for its structure-activity relationship study.

Deep learning driven diagnosis of malignant soft tissue tumors based on dual-modal ultrasound images and clinical indexes.

Background: Soft tissue tumors (STTs) are benign or malignant superficial neoplasms arising from soft tissues throughout the body with versatile pathological types. Although Ultrasonography (US) is one of the most common imaging tools to diagnose malignant STTs, it still has several drawbacks in STT diagnosis that need improving. Objectives: The study aims to establish this deep learning (DL) driven Artificial intelligence (AI) system for predicting malignant STTs based on US images and clinical indexes of the patients. Methods: We retrospectively enrolled 271 malignant and 462 benign masses to build the AI system using 5-fold validation. A prospective dataset of 44 malignant masses and 101 benign masses was used to validate the accuracy of system. A multi-data fusion convolutional neural network, named ultrasound clinical soft tissue tumor net (UC-STTNet), was developed to combine gray scale and color Doppler US images and clinic features for malignant STTs diagnosis. Six radiologists (R1-R6) with three experience levels were invited for reader study. Results: The AI system achieved an area under receiver operating curve (AUC) value of 0.89 in the retrospective dataset. The diagnostic performance of the AI system was higher than that of one of the senior radiologists (AUC of AI vs R2: 0.89 vs. 0.84, p=0.022) and all of the intermediate and junior radiologists (AUC of AI vs R3, R4, R5, R6: 0.89 vs 0.75, 0.81, 0.80, 0.63; p <0.01). The AI system also achieved an AUC of 0.85 in the prospective dataset. With the assistance of the system, the diagnostic performances and inter-observer agreement of the radiologists was improved (AUC of R3, R5, R6: 0.75 to 0.83, 0.80 to 0.85, 0.63 to 0.69; p<0.01). Conclusion: The AI system could be a useful tool in diagnosing malignant STTs, and could also help radiologists improve diagnostic performance.

Genome-wide identification, characterization, molecular evolution and expression profiling analysis of scavenger receptors in black rockfish (Sebastes schlegelii).

The scavenger receptors (SRs) gene family is considered as the membrane-associated pattern recognition receptors that plays important roles in the immune responses of organisms. However, there is currently limited research on the systematic identification of the SRs gene family in teleost and their role in the innate immunity of S. schegelii. In this study, we identified and annotated 15 SRs genes in S. schegelii. Through phylogenetic analysis, analysis of conserved domains, gene structure, and motif composition, we found that SRs gene family within different classes were relatively conserved. Additionally, we used qRT-PCR to analyze the expression patterns of SRs genes in immune-related tissues from healthy and Acinetobacter johnsonii-infected S. schegelii. The results showed that SRs genes exhibited different tissue expression patterns and the expression of SRs genes significantly changed after A. johnsonii infection. These results provided a valuable basis for further understanding of the functions of SRs in the innate immune response of S. schegelii.

KpsS1 Mediates the Glycosylation of Pseudaminic Acid in Acinetobacter Baumannii.

Pseudaminic acid (Pse) is found in the polysaccharide structures of the cell surface of various Gram-negative pathogenic bacteria including Acinetobacter baumannii and considered as an important component of cell surface glycans including oligosaccharides and glycoproteins. However, the glycosyltransferase that is responsible for the Pse glycosylation in A. baumannii remains unknown yet. In this study, through comparative genomics analysis of Pse-positive and negative A. baumannii clinical isolates, we identified a potential glycosyltransferase, KpsS1, located right downstream of the Pse biosynthesis genetic locus. Deletion of this gene in an Pse-positive A. baumannii strain, Ab8, impaired the glycosylation of Pse to the surface CPS and proteins, while the gene knockout strain, Ab8ΔkpsS1, could still produce Pse with 2.86 folds higher amount than that of Ab8. Furthermore, impairment of Pse glycosylation affected the morphology and virulence potential of A. baumannii, suggesting the important role of this protein. This study will provide insights into the further understanding of Pse in bacterial physiology and pathogenesis.

Investigation on the substrate specificity and N-substitution tolerance of PseF in catalytic transformation of pseudaminic acids to CMP-Pse derivatives.

Pseudaminic acid (Pse) belongs to a class of bacterial non-2-ulosonic acids, and has been implicated in bacterial infection and immune evasion. Various Pse structures with diverse N-substitutions have been identified in pathogenic bacterial strains like Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. In this study, we successfully synthesized three new Pse species, including Pse5Ac7Fo, Pse5Ac7(3RHb) and Pse7Fo5(3RHb) using chemical methods. Furthermore, we investigated the substrate specificity of cytidine 5'-monophosphate (CMP)-Pse synthetase (PseF), resulting in the production of N-modified CMP-Pse derivatives (CMP-Pses). It was found that PseF was promiscuous with the Pse substrate and could tolerate different modifications at the two nitrogen atoms. This study provides valuable insights into the incorporation of variable N-substitutions in the Pse biosynthetic pathway.

Real-Time Biosynthetic Reaction Monitoring Informs the Mechanism of Action of Antibiotics.

The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB. We then show how we can inhibit their activities using existing and newly discovered antibiotics such as bacitracin and teixobactin. Additionally, we found that the UppP dimer is stabilized by phosphatidylethanolamine, which, unexpectedly, enhanced the speed of substrate processing. Overall, our results demonstrate the potential of native mass spectrometry for real-time biosynthetic reaction monitoring of membrane enzymes, as well as their in situ inhibition and cofactor binding, to inform the mode of action of emerging antibiotics.

Selective Peptide Cysteine Manipulation on Demand and Difficult Protein Chemical Synthesis Enabled by Controllable Acidolysis of N,S-Benzylidene Thioacetals.

Although solid-phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S-benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V-like (PD-L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.

Discovery of a Monoclonal Antibody That Targets Cell-Surface Pseudaminic Acid of Acinetobacter baumannii with Direct Bactericidal Effect.

The therapeutic effects of antibodies include neutralization of pathogens, activation of the host complement system, and facilitation of phagocytosis of pathogens. However, antibody alone has never been shown to exhibit bactericidal activity. In this study, we developed a monoclonal antibody that targets the bacterial cell surface component Pseudaminic acid (Pse). This monoclonal antibody, Pse-MAB1, exhibited direct bactericidal activity on Acinetobacter baumannii strains, even in the absence of the host complements or other immune factors, and was able to confer a protective effect against A. baumannii infections in mice. This study provides new insight into the potential of developing monoclonal antibody-based antimicrobial therapy of multidrug resistant bacterial infections, especially those which occurred among immunocompromised patients.

Synchronization of inertial complex-valued memristor-based neural networks with time-varying delays.

The synchronization of inertial complex-valued memristor-based neural networks (ICVMNNs) with time-varying delays was explored in the paper with the non-separation and non-reduced approach. Sufficient conditions required for the exponential synchronization of the ICVMNNs were identified with the construction of comprehensive Lyapunov functions and the design of a novel control scheme. The adaptive synchronization was also investigated based on the derived results, which is easier to implement in practice. What's more, a numerical example that verifies the obtained results was presented.

pH-triggered hydrophility-adjustable fluorescent probes for simultaneously imaging lipid droplets and lysosomes and the application in fatty liver detection.

To study the collaboration between lipid droplets (LDs) and lysosomes, and the lipid change in nonalcoholic fatty liver disease (NAFLD), herein two pH-triggered hydrophility-adjustable fluorescent probes (LD-Lyso and LD-Lyso 1) are designed. The mechanism is based on cyclization and ring-opening with thorough consideration of pH and hydrophilic differences between LDs and lysosomes. Both of the two probes exist in ring-opening form and emit red fluorescence in acidic environment, while they exist in cyclized form and the emission is blueshifted in alkaline environment due to reduced conjugate planes. Moreover, LD-Lyso exhibits near infrared fluorescence at 740 nm under ring-opening form, which facilitates further cell, tissue, and in vivo imaging. The cell imaging results show that LD-Lyso can simultaneously target LDs and lysosomes by two different colors. Impressively, LD-Lyso cannot only detect NAFLD tissues from the normal tissue, but also distinguish different degrees of NAFLD tissues and mice, which provides a very promising tool for timely diagnosis of early NAFLD.

A System for Discovering Novel Uricosurics Targeting Urate Transporter 1 Based on In Vitro and In Vivo Modeling.

Hyperuricemia has become a global burden with the increasing prevalence and risk of associated metabolic disorders and cardiovascular diseases. Uricosurics act as a vital urate-lowering therapy by promoting uric acid excretion via the kidneys. However, potent and safe uricosurics are still in urgent demand for use in the clinic. In this study, we aimed to establish in vitro and in vivo models to aid the discovery of novel uricosurics, and to search for potent active compounds, especially targeting urate transporter 1 (URAT1), the major urate transporter in the kidney handling uric acid homeostasis. As a result, for preliminary screening, the in vitro URAT1 transport activity was assessed using a non-isotopic uric acid uptake assay in hURAT1-stably expressed HEK293 cells. The in vivo therapeutic effect was evaluated in a subacute hyperuricemic mouse model (sub-HUA) and further confirmed in a chronic hyperuricemic mouse model (Ch-HUA). By utilizing these models, compound CC18002 was obtained as a potent URAT1 inhibitor, with an IC50 value of 1.69 µM, and favorable uric acid-lowering effect in both sub-HUA and Ch-HUA mice, which was comparable to that of benzbromarone at the same dosage. Moreover, the activity of xanthine oxidoreductase, the key enzyme catalyzing uric acid synthesis, was not altered by CC18002 treatment. Taken together, we have developed a novel screening system, including a cell model targeting URAT1 and two kinds of mouse models, for the discovery of novel uricosurics. Utilizing this system, compound CC18002 was investigated as a candidate URAT1 inhibitor to treat hyperuricemia.

Revealing the extracellular function of HMGB1 N-terminal region acetylation assisted by a protein semi-synthesis approach.

HMGB1 (high-mobility group box 1) is a non-histone chromatin-associated protein that has been widely reported as a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in the proinflammatory process once it is in an extracellular location. Accumulating evidence has shown that HMGB1 undergoes extensive post-translational modifications (PTMs) that actively regulate its conformation, localization, and intermolecular interactions. However, fully characterizing the functional implications of these PTMs has been challenging due to the difficulty in accessing homogeneous HMGB1 with site-specific PTMs of interest. In this study, we developed a streamlined protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation, STL/CPL). This methodology enabled us to generate a series of N-terminal region acetylated HMGB1 proteins. Further studies revealed that acetylation regulates HMGB1-heparin interaction and modulates HMGB1's stability against thrombin, representing a regulatory switch to control HMGB1's extracellular activity.