Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

DNA methylation marker identification and poly-methylation risk score in prediction of healthspan termination.

Aim: To elucidate the epigenetic consequences of DNA methylation in healthspan termination (HST), considering the current limited understanding. Materials & methods: Genetically predicted DNA methylation models were established (n = 2478). These models were applied to genome-wide association study data on HST. Then, a poly-methylation risk score (PMRS) was established in 241,008 individuals from the UK Biobank. Results: Of the 63,046 CpGs from the prediction models, 13 novel CpGs were associated with HST. Furthermore, people with high PMRSs showed higher HST risk (hazard ratio: 1.18; 95% CI: 1.13-1.25). Conclusion: The study indicates that DNA methylation may influence HST by regulating the expression of genes (e.g., PRMT6, CTSK). PMRSs have a promising application in discriminating subpopulations to facilitate early prevention.

The Molecular and Functional Characterization of Sensory Neuron Membrane Protein 1b (SNMP1b) from Cyrtotrachelus buqueti (Coleoptera: Curculionidae).

Sensory neuron membrane proteins (SNMPs) play important roles in insect chemoreception and SNMP1s have been reported to be essential in detecting sex pheromones in Drosophila and some lepidopteran species. However, SNMPs for Cyrtotrachelus buqueti (Coleoptera: Curculionidae), a major insect pest of bamboo plantations, remain uncharacterized. In this study, a novel SNMP gene, CbuqSNMP1b, from C. buqueti was functionally characterized. The expression of CbuqSNMP1b was significantly higher in antennae than in other tissues of both sexes and the expression level was significantly male-biased. Additionally, CbuqSNMP1b showed significantly higher transcription levels in the adult stage and very low transcription levels in other stages, suggesting that CbuqSNMP1b is involved in the process of olfaction. Fluorescence binding assays indicated that CbuqSNMP1b displayed the strongest binding affinity to dibutyl phthalate (Ki = 9.03 µM) followed by benzothiazole (Ki = 11.59 µM) and phenol (Ki = 20.95 µM) among fourteen C. buqueti volatiles. Furthermore, molecular docking revealed key residues in CbuqSNMP1b that interact with dibutyl phthalate, benzothiazole, and phenol. In conclusion, these findings will lay a foundation to further understand the olfactory mechanisms of C. buqueti and promote the development of novel methods for controlling this pest.

Associations between sleep-behavioral traits and healthspan: A one-sample Mendelian randomization study based on 388,909 participants of the UK-Biobank.

BACKGROUND: Although the association between sleep behavior and morbidity and mortality risk has been reported before, there is still uncertainty whether the observed associations are causal or confounding. Therefore, we investigated the causal relationships between sleep-behavioral traits and terminated healthspan risk using Mendelian randomization (MR). METHODS: We conducted a one-sample MR analysis to evaluate causality between six sleep-behavioral traits (sleep duration, chronotype/morningness, napping, sleeplessness/insomnia, and getting up from bed) and risk of healthspan termination among 388, 909 UK Biobank (UKB) participants. Instrumental variables for sleep behaviors (N = 590) were obtained from recent genome-wide association studies (GWAS). We defined healthspan based on eight predominant health-terminating events associated with longevity (congestive heart failure, myocardial infarction, chronic obstructive pulmonary disease, stroke, dementia, diabetes, cancer, and death). We further constructed a sleep score and a weighted genetic risk score to increase the predictive ability of the sleep-behavioral traits. Cox regression models and Inverse Probability Treatment Weighting (IPTW) were implemented, followed by MR to assess causation. We used inverse-variance-weighted MR to estimate causal effects, and weighted-median and MR-egger for sensitivity analysis to test the pleiotropic effects. RESULTS: In IPTW, we observed a decreased risk of terminated healthspan for healthy sleep behaviors such as 'sleep duration 7-8h/d' (Hazard ratio, HR = 0.93; 95 % confidence interval, CI: 0.92-0.96; P < 0.001); 'morningness' (HR = 0.95; 95%CI: 0.93-0.98; P < 0.01); 'napping' (HR = 0.93; 95%CI: 0.91-0.94; P < 0.001); 'easy getting up from bed' (HR = 0.91; 95%CI: 0.88-0.93; P < 0.001); and, 'never/rarely experience sleeplessness/insomnia' (HR = 0.94; 95%CI: 0.92-0.96; P < 0.001). MR results further indicated causal associations between healthy sleep duration (OR = 0.98; 95%CI: 0.97-1.00; P = 0.036) and insomnia (OR = 1.02; 95%CI: 1.01-1.03; P < 0.001) with terminated healthspan. MR-egger did not suggest any potential pleiotropy. CONCLUSION: This study supports abnormal sleep duration and insomnia as potential causal risk factors for terminated healthspan. Thus, healthy sleep behavior is valuable for the extension of healthspan, and well-designed and tailored sleep health interventions are warranted.

Association between sleep traits and biological aging risk: a Mendelian randomization study based on 157 227 cases and 179 332 controls.

STUDY OBJECTIVES: To investigate whether sleep traits are associated with the risk of biological aging using a case-control design with Mendelian randomization (MR) analyses. METHODS: We studied 336 559 participants in the UK Biobank cohort, including 157 227 cases of accelerated biological aging and 179 332 controls. PhenoAge, derived from clinical traits, estimated biological ages, and the discrepancies from chronological age were defined as age accelerations (PhenoAgeAccel). Sleep behaviors were assessed with a standardized questionnaire. propensity score matching matched control participants to age-accelerated participants, and a conditional multivariable logistic regression model estimated odds ratio (OR) and 95% confidence intervals (95% CI). Causal relationships between sleep traits and PhenoAgeAccel were explored using linear and nonlinear MR methods. RESULTS: A U-shaped association was found between sleep duration and PhenoAgeAccel risk. Short sleepers had a 7% higher risk (OR = 1.07; 95% CI: 1.03 to 1.11), while long sleepers had an 18% higher risk (OR = 1.18; 95% CI: 1.15 to 1.22), compared to normal sleepers (6-8 hours/day). Evening chronotype was linked to higher PhenoAgeAccel risk than morning chronotype (OR = 1.14; 95% CI: 1.10 to 1.18), while no significant associations were found for insomnia or snoring. Morning chronotype had a protective effect on PhenoAgeAccel risk (OR = 0.87, 95% CI: 0.79 to 0.95) per linear MR analysis. Genetically predicted sleep duration showed a U-shaped relationship with PhenoAgeAccel, suggesting a nonlinear association (pnonlinear < 0.001). CONCLUSIONS: The study suggests that improving sleep can slow biological aging, highlighting the importance of optimizing sleep as an intervention to mitigate aging's adverse effects.

Effects of Yinzhihuang on Alleviating Cyclosporine A-Induced Cholestatic Liver Injury via Farnesoid X Receptor-Mediated Regulation of Transporters and Enzymes in Vitro and in Vivo.

Yinzhihuang (YZH), a traditional Chinese medicine prescription, was widely used to treat cholestasis. Cholestatic liver injury limited the use of the immunosuppressive drug cyclosporine A (CsA) in preventing organ rejection after solid organ transplantation. Clinical evidences suggested that YZH could enhance bile acids and bilirubin clearance, providing a potential therapeutic strategy against CsA-induced cholestasis. Nevertheless, it remains unclear whether YZH can effectively alleviate CsA-induced cholestatic liver injury, as well as the molecular mechanisms responsible for its hepatoprotective effects. The purpose of the present study was to investigate the hepatoprotective effects of YZH on CsA-induced cholestatic liver injury and explore its molecular mechanisms in vivo and vitro. The results demonstrated that YZH significantly improved the CsA-induced cholestatic liver injury and reduced the level of liver function markers in serum of Sprague-Dawley (SD) rats. Targeted protein and gene analysis indicated that YZH increased bile acids and bilirubin efflux into bile through the regulation of multidrug resistance-associated protein 2 (Mrp2), bile salt export pump (Bsep), sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 2 (Oatp2) transport systems, as well as upstream nuclear receptors farnesoid X receptor (Fxr). Moreover, YZH modulated enzymes involved in bile acids synthesis and bilirubin metabolism including Cyp family 7 subfamily A member 1 (Cyp7a1) and uridine 5'-diphosphate (UDP) glucuronosyltransferase family 1 member A1 (Ugt1a1). Furthermore, the active components geniposidic acid, baicalin and chlorogenic acid exerted regulated metabolic enzymes and transporters in LO2 cells. In conclusion, YZH may prevent CsA-induced cholestasis by regulating the transport systems, metabolic enzymes, and upstream nuclear receptors Fxr to restore bile acid and bilirubin homeostasis. These findings highlight the potential of YZH as a therapeutic intervention for CsA-induced cholestasis and open avenues for further research into its clinical applications.

Stimulus-responsive biomaterials for Helicobacter pylori eradication.

BACKGROUND: Helicobacter pylori (H. pylori), the only bacterium classified as a type I (definite) carcinogen, is strongly associated with the development of gastric inflammation and adenocarcinoma. It infects the stomach of approximately half of the global population, equivalent to nearly 4.4 billion people. However, due to physiological barriers in the stomach, microbial barriers and increased antibiotic resistance, the therapeutic efficiency of standard antibiotic therapy is limited and cannot meet the clinical needs in some areas. Combining stimulus-responsive biomaterials with certain stimuli is an emerging antibacterial strategy. Stimulus-responsive biomaterials can respond to chemical, biological or physical cues in the environment with corresponding changes in their own properties and functions, highlighting a more intelligent, targeting and efficient aspect for H. pylori therapy. AIM OF REVIEW: This review describes the critical obstacles in the current treatment of H. pylori, summarizes the recent advances in stimulus-responsive biomaterials against H. pylori by elucidating their working mechanisms and antibacterial performances under different types of stimuli (pH, enzymes, light, magnetic and ultrasound irradiations), and attempts to analyze the future prospects of such smart biomaterial for H. pylori eradication. Key Scientific Concepts of Review: Any characteristic property or change in the biomilieu at the H. pylori infected site (endogenous stimuli) or specific iatrogenic conditions in vitro (exogenous stimuli) can act as cues to activate or potentiate the antibacterial activity of responsive biomaterials. The responsiveness of these materials to endogenous stimuli enhances antimicrobial targeting, and makes physiological barriers that would otherwise hinder conventional H. pylori therapies a key factor in facilitating antibacterial effects. The responsiveness to exogenous stimuli greatly prolongs the action time of antimicrobial materials and pinpoints the site of infection, thereby reducing toxic side effects. These findings pave the way for the development of more precise and effective anti-H. pylori treatment.

Dimethylaminododecyl Methacrylate-Incorporated Dental Materials Could Be the First Line of Defense against Helicobacter pylori.

Oral cavity is an essential reservoir for H. pylori. We aimed to investigate the antibacterial effects of dimethylaminododecyl methacrylate (DMADDM) against H. pylori. Modified giomers were prepared by introducing 0%, 1.25% and 2.5% DMADDM monomers. Broth microdilution assay, spot assay, Alamer Blue assay, PMA-qPCR, crystal violet staining, scanning electron microscopy observation and live/dead bacterial staining were performed to evaluate the antibacterial and antibiofilm effects of DMADDM and modified giomers in vitro. Urease assay, qPCR, hematoxylin-eosin staining and ELISA were performed to evaluate the inflammation levels and colonization of H. pylori in vivo. In vitro experiments indicated that the minimum inhibitory concentration and minimum bactericidal concentration of DMADDM were 6.25 µg/mL and 25 µg/mL, respectively. It inhibited H. pylori in a dose- and time-dependent manner, and significantly reduced the expression of cagA, vacA, flaA and ureB. DMADDM-modified giomers inhibited the formation of H. pylori biofilm and reduced live cells within it. In vivo experiments confirmed that the pretreatment with DMADDM-modified dental resin effectively reduced the gastric colonization of oral-derived H. pylori, suppressed systemic and local gastric inflammation. DMADDM monomers and DMADDM-modified giomers possessed excellent antibacterial and antibiofilm effects on H. pylori. Pretreatment with DMADDM-modified giomers significantly inhibited the gastric infection by H. pylori.

Complete genome sequence of Brucella pseudintermedia ASAG-D25, isolated from the wheat ear.

Brucella pseudintermedia ASAG-D25 was isolated from the wheat ear sample in Xuzhou City, Jiangsu Province, China. The complete genome sequence of B. pseudintermedia will provide an important resource for better understanding of the genetic features of the species within the family of Brucellaceae.

Polygenic risk for termination of the 'healthspan' and its interactions with lifestyle factors: A prospective cohort study based on 288,359 participants.

OBJECTIVES: To investigate whether a polygenic risk score (PRS) and its interactions with lifestyle factors are associated with termination of the 'healthspan' (the number of years living without serious diseases or degeneration). DESIGN, EXPOSURES AND PARTICIPANTS: Death or the incidence of any of seven independent morbidities (cancer, congestive heart failure, myocardial infarction, chronic obstructive pulmonary disease, stroke, dementia, and diabetes) strongly associated with aging were considered to define the termination of the healthspan. A total of 288,359 healthy participants from the UK Biobank were included in this prospective cohort study to evaluate the associations between PRS, lifestyle, and healthspan. The PRS was generated by weighting 12 healthspan-related genetic loci, which and scores were then categorized into three groups in Cox regression models. A lifestyle index was developed that incorporated body mass index (BMI), alcohol consumption, diet, smoking, and physical activity, and these scores were also categorized into three groups. The risk of termination of the healthspan was calculated across the different PRS and lifestyle index groups using Cox regression models. Interactions were estimated with the marginal effect of lifestyle on the risk of termination of healthspan across values of the moderator PRS using kernel estimation. RESULTS: During an average follow-up of 9.83 years, 68,903 healthspan-termination events occurred. It was calculated that people with high polygenic risk could reduce their risk of healthspan termination by 40 % if they maintain a favorable lifestyle. The marginal effect of lifestyle on the risk of healthspan termination increased with growing genetic risk. Smoking and diet showed monotonic changes in opposite directions, while BMI, physical activity, and alcohol had a U-shaped interaction with genetic risk. CONCLUSIONS: Favorable lifestyle can attenuate the risk of termination of the healthspan, especially for people with high genetic risk. The improvement afforded by ideal lifestyle behaviors varies for each individual.

A parylene-mediated plasmonic-photonic hybrid fiber-optic sensor and its instrumentation for miniaturized and self-referenced biosensing.

With the development of advanced nanofabrication techniques over the past decades, different nanostructure-based plasmonic fiber-optic sensors have been developed and have presented a low limit of detection for various biomolecules. However, owing to both the dependence on complex equipment and the trade-off between the fabrication cost and sensing performance, nanostructured plasmonic fiber-optic sensors are rarely used outside laboratories. To facilitate wider application of the plasmonic fiber-optic sensors, a parylene-mediated hybrid plasmonic-photonic cavity-based sensor was developed. Compared with a similar plasmonic sensor which only works in the plasmonic mode, the proposed hybrid sensor shows a higher reproducibility (CV < 2.5%) due to its resistance to fabrication variations. Meanwhile, a self-referenced detection mechanism and a novel miniaturized system were developed to adapt to the hybrid resonance sensor. The entire system only has a weight of 263 g, and a size of 12 cm × 10 cm × 8 cm, and is especially suitable for outdoor applications in a handheld manner. In experiments, a high refractive index sensitivity of 3.148 RIU-1 and real-time biomolecule monitoring at nanomolar concentrations were achieved by the proposed system, further confirming the potential of the miniaturized system as a candidate for point-of-care health diagnostics outside laboratories.

Oxidation-Induced Protein Cross-Linking in Mammalian Cells.

A proximity-enabled protein cross-linking strategy with additional spatiotemporal control is highly desirable. Here, we report an oxidation-induced protein cross-linking strategy involving the incorporation of a vinyl thioether group into proteins in both Escherichia coli and mammalian cells via genetic code expansion. We demonstrated that vinyl thioether can be selectively induced by exogenously added oxidant or by reactive oxygen species from the cellular environment, as well as by photocatalysts, and converted into a Michael acceptor, enabling fluorescence labeling and protein cross-linking.

Genetically encoded chemical crosslinking of carbohydrate.

Protein-carbohydrate interactions play important roles in various biological processes, such as organism development, cancer metastasis, pathogen infection and immune response, but they remain challenging to study and exploit due to their low binding affinity and non-covalent nature. Here we site-specifically engineered covalent linkages between proteins and carbohydrates under biocompatible conditions. We show that sulfonyl fluoride reacts with glycans via a proximity-enabled reactivity, and to harness this a bioreactive unnatural amino acid (SFY) that contains sulfonyl fluoride was genetically encoded into proteins. SFY-incorporated Siglec-7 crosslinked with its sialoglycan ligand specifically in vitro and on the surface of cancer cells. Through irreversible cloaking of sialoglycan at the cancer cell surface, SFY-incorporated Siglec-7 enhanced the killing of cancer cells by natural killer cells. Genetically encoding the chemical crosslinking of proteins to carbohydrates (GECX-sugar) offers a solution to address the low affinity and weak strength of protein-sugar interactions.

Genetically encoded chemical crosslinking of RNA in vivo.

Protein-RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein-RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.

Application of information-intelligence technologies in pharmacy intravenous admixture services in a Chinese third-class a hospital.

BACKGROUND: Pharmacy intravenous admixture service (PIVAS) center has emerged as an important department of hospital as it can improve occupational protection and ensure the safety and effectiveness of intravenous infusions. However, medication errors were considered to be a significant challenge in PIVAS, so information-intelligence technologies were introduced to optimize the management of PIVAS. Our article summarized the application of information-intelligence technologies in PIVAS of a large third-class A hospital in China, and provided an example for PIVAS in other hospitals at home and abroad. METHODS: Prescription-reviewing rules containing intravenous medications and infusion solution guideline were recorded in the database of prescription-cheking system. Drugs information were recorded in the PIVAS management system with special identification and warning labels to reduce intravenous infusion errors. Automatic labeling device was used to label the infusion bags, and the quality control program database of intelligent compounding robot for cytotoxic drugs was established ingeniously. Automatic sorting devices were applied for the third batch of finished infusion admixtures, and intelligent logistics robots were used to transport the infusion to the ward. RESULTS: After establishing and implementing of prescription-reviewing rules in the prescription-cheking system database, the number of prescriptions checked by pharmacists increased from 18 to 43 per minute. The success rate of intervention with irrational medical orders increased from 85.89% to 99.06% (P < 0.05). By introducing various intelligent devices, automatic labeling significantly enhanced work efficiency and reduced the error rate (P < 0.001). Furthermore, the use of intelligent intravenous compounding robots significantly reduced the risk of errors (P < 0.001). CONCLUSIONS: The application of information-intelligence technologies in PIVAS can improve work efficiency and reduce error risk. However, some intelligent devices have failed to achieve the expected effect in practical use, and further improvements are needed to meet the demands of PIVAS in the future.

Analyses of rare predisposing variants of lung cancer in 6,004 whole genomes in Chinese.

We present the largest whole-genome sequencing (WGS) study of non-small cell lung cancer (NSCLC) to date among 6,004 individuals of Chinese ancestry, coupled with 23,049 individuals genotyped by SNP array. We construct a high-quality haplotype reference panel for imputation and identify 20 common and low-frequency loci (minor allele frequency [MAF] ≥ 0.5%), including five loci that have never been reported before. For rare loss-of-function (LoF) variants (MAF < 0.5%), we identify BRCA2 and 18 other cancer predisposition genes that affect 5.29% of individuals with NSCLC, and 98.91% (181 of 183) of LoF variants have not been linked previously to NSCLC risk. Promoter variants of BRCA2 also have a substantial effect on NSCLC risk, and their prevalence is comparable with BRCA2 LoF variants. The associations are validated in an independent case-control study including 4,410 individuals and a prospective cohort study including 23,826 individuals. Our findings not only provide a high-quality reference panel for future array-based association studies but depict the whole picture of rare pathogenic variants for NSCLC.

The interactions between oral-gut axis microbiota and Helicobacter pylori.

In the human body, each microbial habitat exhibits a different microbial population pattern, and these distinctive microflorae are highly related to the development of diseases. The microbial interactions from host different niches are becoming crucial regulators to shape the microbiota and their physiological or pathological functions. The oral cavity and gut are the most complex and interdependent microbial habitats. Helicobacter pylori is one of the most important pathogens from digestive tract, especially the stomach, due to its direct relationships with many gastric diseases including gastric cancer. H. pylori infections can destroy the normal gastric environment and make the stomach a livable channel to enhance the microbial interactions between oral cavity and gut, thus reshaping the oral and gut microbiomes. H. pylori can be also detected in the oral and gut, while the interaction between the oral-gut axis microbiota and H. pylori plays a major role in H. pylori's colonization, infection, and pathogenicity. Both the infection and eradication of H. pylori and its interaction with oral-gut axis microbiota can alter the balance of the microecology of the oral-gut axis, which can affect the occurrence and progress of related diseases. The shift of oral-gut axis microbiota and their interactions with H. pylori maybe potential targets for H. pylori infectious diagnosis and treatment.

Accelerating PERx reaction enables covalent nanobodies for potent neutralization of SARS-CoV-2 and variants.

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity, yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here, we have developed covalent nanobodies capable of binding with SARS-CoV-2 irreversibly via a proximity-enabled reactive therapeutic (PERx) mechanism. A latent bioreactive amino acid (FFY) was designed and genetically encoded into nanobodies to accelerate the PERx reaction rate. Compared with the noncovalent wild-type nanobody, the FFY-incorporated covalent nanobodies neutralized both wild-type SARS-CoV-2 and its Alpha, Delta, Epsilon, Lambda, and Omicron variants with drastically higher potency. This PERx-enabled covalent-nanobody strategy and the related insights into increased potency can be valuable to developing effective therapeutics for various viral infections.

Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

Single-cell profiling of human CD127+ innate lymphoid cells reveals diverse immune phenotypes in hepatocellular carcinoma.

BACKGROUND AND AIMS: Innate lymphoid cells (ILCs) are tissue-resident lymphocytes that play critical roles in cytokine-mediated regulation of homeostasis and inflammation. However, relationships between their immune phenotypic characteristics and HCC remain largely unexplored. APPROACH AND RESULTS: We performed single-cell RNA sequencing analysis on sorted hepatic ILC cells from human patients with HCC and validated using flow cytometry, multiplex immunofluorescence staining, and functional experiments. Moreover, we applied selection strategies to enrich ILC populations in HCC samples to investigate the effects of B cells on the immune reaction of inducible T cell costimulator (ICOS)+ ILC2 cells. Dysregulation of ILCs was manifested by the changes in cell numbers or subset proportions in HCC. Seven subsets of 3433 ILCs were identified with unique properties, of which ICOS+ ILC2a were preferentially enriched in HCC and correlated with poor prognosis. Mechanistically, we report that B cells, particularly resting naïve B cells, have a previously unrecognized function that is involved in inflammatory differentiation of ILC2 cells. B cell-derived ICOSL signaling was responsible for exacerbating inflammation through the increased production of IL-13 in ICOS+ ILC2a cells. Heat shock protein 70 (HSP70) genes Heat Shock Protein Family A Member 1A (HSPA1A) and Heat Shock Protein Family A Member 1B (HSPA1B) were highly expressed in ILC2s in late-stage HCC, and targeting to ICOS and its downstream effector HSP70 in ILC2s suppressed tumor growth and remodeled the immunosuppressive tumor microenvironment. CONCLUSIONS: This in-depth understanding sheds light on B cell-driven innate type 2 inflammation and provides a potential strategy for HCC immunotherapy.