IgG sialylation occurs in B cells pre antibody secretion.

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.

Automated high throughput IgG N-glycosylation sample preparation method development on the Tecan Freedom EVO platform.

Introduction: Glycomics, focusing on the role of glycans in biological processes, particularly their influence on the folding, stability and receptor interactions of glycoconjugates like antibodies, is vital for our understanding of biology. Changes in immunoglobulin G (IgG) N-glycosylation have been associated with various physiological and pathophysiological conditions. Nevertheless, time-consuming manual sample preparation is one of the limitations in the glycomics diagnostic implementation. The study aimed to develop an automated method for sample preparation on the Tecan Freedom Evo 200 platform and compare its efficiency and precision with the manual counterpart. Materials and methods: The initial method development included 32 pooled blood plasma technical replicates. An additional 24 pooled samples were used in the method comparison along with 78 random duplicates of plasma samples collected from 10,001 Dalmatians biobank to compare the manual and automated methods. Results: The development resulted in a new automated method. For the automated method, glycan peaks comprising 91% of the total sample glycan showed a variation of less than 5% while 92% of the total sample showed a variation of less than 5% for the manual method. The results of the Passing-Bablok regression indicated no differences between the automated and manual methods for 12 glycan peaks (GPs). However, for 8 GPs systematic difference was present, while both systematic and proportional differences were present for four GPs. Conclusions: The developed automated sample preparation method for IgG glycan analysis reduced exposure to hazardous chemicals and offered a simplified workflow. Despite slight differences between the methods, the new automated method showed high precision and proved to be highly comparable to its manual counterpart.

Glycan clock of ageing-analytical precision and time-dependent inter- and i-individual variability.

Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age.For precision analysis, three different plasma pools were analysed over 26 days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3 months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10 years, respectively. Samples were collected every 3 months and 3 weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits.The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits.The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths.

Variability of human Alpha-1-acid glycoprotein N-glycome in a Caucasian population.

AIM: Alpha-1-acid glycoprotein (AGP) is a highly glycosylated protein in human plasma and one of the most abundant acute phase proteins in humans. Glycosylation plays a crucial role in its biological functions, and alterations in AGP N-glycome have been associated with various diseases and inflammatory conditions. However, large-scale studies of AGP N-glycosylation in the general population are lacking. METHODS: Using recently developed high-throughput glycoproteomic workflow for site-specific AGP N-glycosylation analysis, 803 individuals from the Croatian island of Korcula were analyzed and their AGP N-glycome data associated with biochemical and physiological traits, as well as different environmental factors. RESULTS: After regression analysis, we found that AGP N-glycosylation is strongly associated with sex, somewhat less with age, along with multiple biochemical and physiological traits (e.g. BMI, triglycerides, uric acid, glucose, smoking status, fibrinogen). CONCLUSION: For the first time we have extensively explored the inter-individual variability of AGP N-glycome in a general human population, demonstrating its changes with sex, age, biochemical, and physiological status of individuals, providing the baseline for future population and clinical studies.

Functional Signature of LRP4 Antibodies in Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Antibodies (Abs) specific for the low-density lipoprotein receptor-related protein 4 (LRP4) occur in up to 5% of patients with myasthenia gravis (MG). The objective of this study was to profile LRP4-Ab effector actions. METHODS: We evaluated the efficacy of LRP4-specific compared with AChR-specific IgG to induce Ab-dependent cellular phagocytosis (ADCP), Ab-dependent cellular cytotoxicity (ADCC), and Ab-dependent complement deposition (ADCD). Functional features were additionally assessed in an independent AChR-Ab+ MG cohort. Levels of circulating activated complement proteins and frequency of Fc glycovariants were quantified and compared with demographically matched 19 healthy controls. RESULTS: Effector actions that required binding of Fc domains to cellular FcRs such as ADCC and ADCP were detectable for both LRP4-specific and AChR-specific Abs. In contrast to AChR-Abs, LRP4-binding Abs showed poor efficacy in inducing complement deposition. Levels of circulating activated complement proteins were not substantially increased in LRP4-Ab-positive MG. Frequency of IgG glycovariants carrying 2 sialic acid residues, indicative for anti-inflammatory IgG activity, was decreased in patients with LRP4-Ab-positive MG. DISCUSSION: LRP4-Abs are more effective in inducing cellular FcR-mediated effector mechanisms than Ab-dependent complement activation. Their functional signature is different from AChR-specific Abs.

Machine learning framework to extract the biomarker potential of plasma IgG N-glycans towards disease risk stratification.

Effective management of chronic diseases and cancer can greatly benefit from disease-specific biomarkers that enable informative screening and timely diagnosis. IgG N-glycans found in human plasma have the potential to be minimally invasive disease-specific biomarkers for all stages of disease development due to their plasticity in response to various genetic and environmental stimuli. Data analysis and machine learning (ML) approaches can assist in harnessing the potential of IgG glycomics towards biomarker discovery and the development of reliable predictive tools for disease screening. This study proposes an ML-based N-glycomic analysis framework that can be employed to build, optimise, and evaluate multiple ML pipelines to stratify patients based on disease risk in an interpretable manner. To design and test this framework, a published colorectal cancer (CRC) dataset from the Study of Colorectal Cancer in Scotland (SOCCS) cohort (1999-2006) was used. In particular, among the different pipelines tested, an XGBoost-based ML pipeline, which was tuned using multi-objective optimisation, calibrated using an inductive Venn-Abers predictor (IVAP), and evaluated via a nested cross-validation (NCV) scheme, achieved a mean area under the Receiver Operating Characteristic Curve (AUC-ROC) of 0.771 when classifying between age-, and sex-matched healthy controls and CRC patients. This performance suggests the potential of using the relative abundance of IgG N-glycans to define populations at elevated CRC risk who merit investigation or surveillance. Finally, the IgG N-glycans that highly impact CRC classification decisions were identified using a global model-agnostic interpretability technique, namely Accumulated Local Effects (ALE). We envision that open-source computational frameworks, such as the one presented herein, will be useful in supporting the translation of glycan-based biomarkers into clinical applications.

N-Glycosylation Profiles of Immunoglobulin G and Future Cardiovascular Events.

BACKGROUND: Posttranslational glycosylation of IgG can modulate its inflammatory capacity through structural variations. We examined the association of baseline IgG N-glycans and an IgG glycan score with incident cardiovascular disease (CVD). METHODS: IgG N-glycans were measured in 2 nested CVD case-control studies: JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin; NCT00239681; primary prevention; discovery; Npairs=162); and TNT trial (Treating to New Targets; NCT00327691; secondary prevention; validation; Npairs=397). Using conditional logistic regression, we investigated the association of future CVD with baseline IgG N-glycans and a glycan score adjusting for clinical risk factors (statin treatment, age, sex, race, lipids, hypertension, and smoking) in JUPITER. Significant associations were validated in TNT, using a similar model further adjusted for diabetes. Using least absolute shrinkage and selection operator regression, an IgG glycan score was derived in JUPITER as a linear combination of selected IgG N-glycans. RESULTS: Six IgG N-glycans were associated with CVD in both studies: an agalactosylated glycan (IgG-GP4) was positively associated, while 3 digalactosylated glycans (IgG glycan peaks 12, 13, 14) and 2 monosialylated glycans (IgG glycan peaks 18, 20) were negatively associated with CVD after multiple testing correction (overall false discovery rate <0.05). Four selected IgG N-glycans comprised the IgG glycan score, which was associated with CVD in JUPITER (adjusted hazard ratio per glycan score SD, 2.08 [95% CI, 1.52-2.84]) and validated in TNT (adjusted hazard ratio per SD, 1.20 [95% CI, 1.03-1.39]). The area under the curve changed from 0.693 for the model without the score to 0.728 with the score in JUPITER (PLRT=1.1×10-6) and from 0.635 to 0.637 in TNT (PLRT=0.017). CONCLUSIONS: An IgG N-glycan profile was associated with incident CVD in 2 populations (primary and secondary prevention), involving an agalactosylated glycan associated with increased risk of CVD, while several digalactosylated and sialylated IgG glycans associated with decreased risk. An IgG glycan score was positively associated with future CVD.

Alterations in plasma protein N-glycosylation after caloric restriction and bariatric surgery.

BACKGROUND: Protein glycosylation is an enzymatic process known to reflect an individual's physiologic state and changes thereof. The impact of metabolic interventions on plasma protein N-glycosylation has only been sparsely investigated. OBJECTIVE: To examine alterations in plasma protein N-glycosylation following changes in caloric intake and bariatric surgery. SETTING: University of Texas Southwestern Medical Center, US and Oxford University Hospitals, UK. METHODS: This study included 2 independent patient cohorts that recruited 10 and 37 individuals with obesity undergoing a period of caloric restriction followed by bariatric surgery. In both cohorts, clinical data were collated, and the composition of plasma protein N-glycome was analyzed chromatographically. Linear mixed models adjusting for age, sex, and multiple testing (false discovery rate <.05) were used to investigate longitudinal changes in glycosylation features and metabolic clinical markers. RESULTS: A low-calorie diet resulted in a decrease in high-branched trigalactosylated and trisialylated plasma N-glycans and a concomitant increase in low-branched N-glycans in both cohorts. Participants from one cohort additionally underwent a washout period during which caloric intake and body weight increased, resulting in reversal of the initial low-calorie diet-related changes in the plasma N-glycome. Immediate postoperative follow-up revealed the same pattern of N-glycosylation changes in both cohorts-an increase in complex, high-branched, antennary fucosylated, extensively galactosylated and sialylated N-glycans and a substantial decline in simpler, low-branched, core fucosylated, bisected, agalactosylated, and asialylated glycans. A 12-month postoperative monitoring in one cohort showed that N-glycan complexity declines while low branching increases. CONCLUSIONS: Plasma protein N-glycosylation undergoes extensive alterations following caloric restriction and bariatric surgery. These comprehensive changes may reflect the varying inflammatory status of the individual following dietary and surgical interventions and subsequent weight loss.

Anastrozole and Tamoxifen Impact on IgG Glycome Composition Dynamics in Luminal A and Luminal B Breast Cancers.

This study examines the intricate relationship between protein glycosylation dynamics and therapeutic responses in Luminal A and Luminal B breast cancer subtypes, focusing on anastrozole and tamoxifen impacts. The present methods inadequately monitor and forecast patient reactions to these treatments, leaving individuals vulnerable to the potential adverse effects of these medications. This research investigated glycan structural changes by following patients for up to 9 months. The protocol involved a series of automated steps including IgG isolation, protein denaturation, glycan labelling, purification, and final analysis using capillary gel electrophoresis with laser-induced fluorescence. The results suggested the significant role of glycan modifications in breast cancer progression, revealing distinctive trends in how anastrozole and tamoxifen elicit varied responses. The findings indicate anastrozole's association with reduced sialylation and increased core fucosylation, while tamoxifen correlated with increased sialylation and decreased core fucosylation. These observations suggest potential immunomodulatory effects: anastrozole possibly reducing inflammation and tamoxifen impacting immune-mediated cytotoxicity. This study strongly emphasizes the importance of considering specific glycan traits to comprehend the dynamic mechanisms driving breast cancer progression and the effects of targeted therapies. The nuanced differences observed in glycan modifications between these two treatments underscore the necessity for further comprehensive research aimed at thoroughly evaluating the long-term implications and therapeutic efficacy for breast cancer patients.

Influence of Desialylation on the Drug Binding Affinity of Human Alpha-1-Acid Glycoprotein Assessed by Microscale Thermophoresis.

Human serum alpha-1-acid glycoprotein (AAG) is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. The sialic acid groups that terminate the N-glycan chains of AAG have been reported to change in response to numerous health conditions and may have an impact on the binding of drugs to AAG. In this study, we quantified the binding between native and desialylated AAG and seven drugs from different pharmacotherapeutic groups (carvedilol, diltiazem, dipyridamole, imipramine, lidocaine, propranolol, vinblastine) using microscale thermophoresis (MST). This method was chosen due to its robustness and high sensitivity, allowing precise quantification of molecular interactions based on the thermophoretic movement of fluorescent molecules. Detailed glycan analysis of native and desialylated AAG showed over 98% reduction in sialic acid content for the enzymatically desialylated AAG. The MST results indicate that desialylation generally alters the binding affinity between AAG and drugs, leading to either an increase or decrease in Kd values, probably due to conformational changes of AAG caused by the different sialic acid content. This effect is also reflected in an increased denaturation temperature of desialylated AAG. Our findings indicate that desialylation impacts free drug concentrations differently, depending on the binding affinity of the drug with AAG relative to human serum albumin (HSA). For drugs such as dipyridamole, lidocaine, and carvedilol, which have a higher affinity for AAG, desialylation significantly changes free drug concentrations. In contrast, drugs such as propranolol, imipramine, and vinblastine, which have a strong albumin binding, show only minimal changes. It is noteworthy that the free drug concentration of dipyridamole is particularly sensitive to changes in AAG concentration and glycosylation, with a decrease of up to 15% being observed, underscoring the need for dosage adjustments in personalized medicine.

MMP3 single-nucleotide polymorphisms are associated with noncontact ACL injuries in competing high-level athletes.

Matrix metalloproteinases (MMPs) play an important role in matrix remodeling, as well as in ligament integrity. Anterior cruciate ligament (ACL) rupture is a severe and frequent knee injury in sports. The aim of this study was to investigate polymorphisms within the MMP3 gene with the predisposition for noncontact ACL rupture in the Croatian professional athletes. One hundred eighty-seven (95 with ACL rupture occurring through a noncontact mechanism and 92 asymptomatic controls) unrelated Caucasians were recruited between 2016 and 2017. All participants were genotyped for three single-nucleotide polymorphisms (SNP) within the MMP3 gene: rs591058 C/T, rs650108 A/G, and rs679620 G/A using the pyrosequencing method. For all three investigated SNPs, genotype frequencies have significantly differed between cases and controls. The MMP3 rs591058 TT (p = 0.0012, odds ratio [OR] = 38.541, 95% confidence interval [CI] = 1.7024-8.7254), rs650108 GG (p = 0.0051, OR = 23.338, 95% CI = 1.2899-4.2226) and rs679620 AA (p = 0.0030, OR = 34.750, 95% CI = 1.5266-7.9101) genotypes, as well as haplotype variant T-G-A (p = 0.0104, OR = 1.71, 95% CI = 1.13-2.59) were significantly overrepresented in cases compared to controls. These results support association between functional variants within the MMP3 gene and the risk of ACL rupture. Still, further research is needed to corroborate these results in a larger population.

Reversal of biological age in multiple rat organs by young porcine plasma fraction.

Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young adult pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n = 613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain, liver, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n = 1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers, behavioral responses encompassing cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro- to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.

Regular moderate physical exercise decreases Glycan Age index of biological age and reduces inflammatory potential of Immunoglobulin G.

Physical inactivity and obesity are growing concerns, negatively impacting the general population. Moderate physical activity is known to have a beneficial anti-inflammatory effect. N-glycosylation of immunoglobulin G (IgG) reflects changes in the inflammatory potential of IgG. In this study, GlycanAge index of biological age (GlycanAge), one of the first commercially used biomarkers of aging, was employed to assess effects of exercise intensity in three different groups of athletes: professional competing athletes, regularly moderate active individuals and newly involved recreational individuals, compared to the group of inactive individuals. GlycanAge was significantly lower in the active group compared to the inactive group (ß = -7.437, p.adj = 7.85E-03), and nominally significant and increased in professional athletes compared to the active group (ß = 7.546, p = 3.20E-02). Competing female athletes had significantly higher GlycanAge comparing to active females exercising moderately (ß = 20.206, p.adj = 2.71E-02), while the latter had significantly lower GlycanAge when compared with the inactive counterparts (ß = -9.762, p.adj = 4.68E-02). Regular, life-long moderate exercise has an anti-inflammatory effect in both female and male population, demonstrated by lower GlycanAge index, and it has great potential to mitigate growing issues related to obesity and a sedentary lifestyle, which are relentlessly increasing world-wide.

Human-specific features and developmental dynamics of the brain N-glycome.

Comparative "omics" studies have revealed unique aspects of human neurobiology, yet an evolutionary perspective of the brain N-glycome is lacking. We performed multiregional characterization of rat, macaque, chimpanzee, and human brain N-glycomes using chromatography and mass spectrometry and then integrated these data with complementary glycotranscriptomic data. We found that, in primates, the brain N-glycome has diverged more rapidly than the underlying transcriptomic framework, providing a means for rapidly generating additional interspecies diversity. Our data suggest that brain N-glycome evolution in hominids has been characterized by an overall increase in complexity coupled with a shift toward increased usage of α(2-6)-linked N-acetylneuraminic acid. Moreover, interspecies differences in the cell type expression pattern of key glycogenes were identified, including some human-specific differences, which may underpin this evolutionary divergence. Last, by comparing the prenatal and adult human brain N-glycomes, we uncovered region-specific neurodevelopmental pathways that lead to distinct spatial N-glycosylation profiles in the mature brain.

Mapping of the gene network that regulates glycan clock of ageing.

Glycans are an essential structural component of immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated. One of the key functional features of IgG glycosylation is the addition of galactose (galactosylation), a trait which was shown to be associated with ageing. We performed GWAS of IgG galactosylation (N=13,705) and identified 16 significantly associated loci, indicating that IgG galactosylation is regulated by a complex network of genes that extends beyond the galactosyltransferase enzyme that adds galactose to IgG glycans. Gene prioritization identified 37 candidate genes. Using a recently developed CRISPR/dCas9 system we manipulated gene expression of candidate genes in the in vitro IgG expression system. Upregulation of three genes, EEF1A1, MANBA and TNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in this in vitro expression system.

Can we suppress chronic systemic inflammation and postpone age-related diseases by targeting the IgG glycome?

INTRODUCTION: Glycans attached to immunoglobulin G are an important regulator of chronic systemic inflammation, one of the key drivers of aging. As people age, glycans that suppress inflammation are being replaced with inflammation-promoting glycans, but the rate of this conversion is highly individual and is affected by genetic, epigenetic, and environmental factors. AREAS COVERED: This review summarizes key studies of IgG glycosylation changes in aging and disease, effects of lifestyle and pharmacological interventions, and mechanisms that regulate IgG glycosylation. EXPERT OPINION: IgG glycome is an important contributor to the process of aging that can be modulated by both lifestyle and pharmacological interventions. Small molecule drugs that would suppress chronic systemic inflammation by modulation of the IgG glycome are still not available, but since gene network regulating IgG glycosylation has been identified and a high-throughput in vitro screening system is available, it is likely that this highly innovative approach to manage chronic systemic inflammation will be developed soon.

Reversal of Biological Age in Multiple Rat Organs by Young Porcine Plasma Fraction.

Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n=613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain-, liver-, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n=1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro- to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.