Genos (Croatia)

A bstract Recruitment and resolution of immune cell accumulation after myocardial infarction (MI) is critical for effective wound healing and prevention of maladaptive remodeling. Numerous signals are known to recruit neutrophils, critical drivers of inflammation, but knowledge about signals containing and resolving their accumulation is limited. We discovered that Regenerating-islet derived protein 3 beta (REG3β) limits the persistence of neutrophils after MI, thereby promoting resolution of inflammation. REG3β selectively binds to aged and hyperactive neutrophils and induces rapid cell death, enabling clearance via macrophage-mediated efferocytosis. Selective binding of REG3β is achieved by interaction with paucimannosylated proteins that translocate from azurophile granules to the plasma membrane in an activation- and age-dependent manner. Endocytotic uptake and accumulation of REG3β in lysosomes initiates programmed cell death of neutrophils via lyosomal membrane permeabilization and release of cathepsins. Our work establishes REG3β as a local immune checkpoint essential for neutrophil resolution and cardiac repair.

The genetic regulation of the plasma N-glycome variation in human populations is not fully characterized, partly due to the limited sample size in glyco-genetics studies. Here, we aimed to demonstrate that protein-specific N-glycan profiles, like those of immunoglobulin G (IgG), can be accurately reconstructed from the total plasma N-glycome (TPNG), enabling us to find new regulators of this complex process re-analysing existing datasets. By testing multiple linear and non-linear machine learning approaches we built a model to reconstruct IgG N-glycans from TPNG data, training on the TwinsUK cohort and validating on CEDAR. We reconstruct GWAS summary statistics for IgG N-glycans by applying the trained linear model to plasma glycan GWAS summary statistics, i.e., as GWAS of linear combinations of plasma glycan traits. The majority of the identified loci had been implicated in IgG N-glycosylation GWAS. Additionally, we found four new loci and suggested the role of FCRLA, KDELR2, HHEX, and TCF3 in the regulation of IgG N-glycosylation. In conclusion, we showed that our method enables the creation of protein-specific N-glycome datasets, allowing for powerful meta-analyses without the need to profile new samples.

Background/Objectives: Southeastern Europe and Croatia have served as a genetic crossroads between the Near East and Europe since prehistoric times, shaped by numerous and repeated migrations. By integrating 19 newly generated ancient genomes with 285 previously published ancient genomes from Croatia, we investigated patterns of maternal and paternal landscapes from the Neolithic, Bronze, and Iron Ages through to the Antiquity and medieval periods, as well as the modern Croatian population. Methods: Ancient DNA extraction from human remains and library preparation were conducted in dedicated clean-room facilities, followed by high-throughput sequencing on the Illumina platform. Sequencing data were analyzed with established pipelines to determine mitochondrial and Y-chromosomal haplogroups and the genetic sex of individuals. Results: New ancient data reveal a predominantly European maternal profile, dominated by haplogroups H, U, and HV0, whereas Y-chromosomal lineages are characterized by J subclades and R1a, with limited representation of R1b and the absence of I2a. When combined with published ancient Croatian genomes, the results reveal similar haplogroup diversity and patterns, as well as the expansion of mtDNA haplogroup H over time and a substantial increase in Y-chromosome R1a and I2a haplogroup frequency from the prehistoric to the modern period. Conclusions: Although the analyzed samples are heterogeneous and originate from different historical periods, their genetic signatures conform to the broader patterns expected for the region. In a wider context, the ancient Croatian mitochondrial data reveal stronger genetic persistence from prehistory to modern times, unlike paternal lineages, which show significantly higher divergence.

Abstract Background and Aims ANCA-associated vasculitis (AAV) activity during and after the treatment lacks adequate biomarkers. Immunoglobuline G (IgG) N-glycome is an enzymatically attached oligosaccharides network crucial for IgG functional “phenotype”. Glicosylation is necessary in inflammatory reactions in which changes occur to glycosylation of numerous plasma proteins including IgG. Recently published paper by Wojcik I et al. pointed to specific IgG glycosylation differences preceding relapse in PR3-AAV patients both with and without ANCA titre rise. By presenting the pilot results of our GlycANVasc (Glycans in ANCA-associated vasculitis) study, evaluating IgG N-glycome changes during the immunosuppressive treatment, we wish to invite collaborators to join in. Method The pilot cases were two female patients, aged 63 (MPO-AAV) and 67 (PR3-AAV), both with lung and renal involvement. Serum was collected at four-time points: The first and second points (both patients) were the at the induction treatment (cyclophosphamide + methylprednisolone) and remission achievement. The third point (only the first patient) was maintenance treatment Rituximab (RTX) administration (three months later), and the fourth point (only the first patient) was one month after RTX. N-glycans were enzymatically released from isolated IgG, fluorophore-labeled, separated using capillary gel electrophoresis, and the glycan traits were calculated using the individual glycans' relative abundances. Results During induction treatment in the “MPO-AAV” patient, the relevant relative changes were the increase in digalactosylated glycans (12.2%) and the decrease in agalactosylated (8.5%) and afucosylated (12.3%). An analogous pattern was present between the second and third samples. Still, the changes were lower: digalactosylated glycans went up by 3.8%, with a reduction of 4.4% in agalactosylated and 2.0% in afucosylated traits. A month after the RTX administration, the relative decreases in the disialylated (8.8%) and monosialylated (3.8%) glycans appeared, while the abundance of (a)galactosylated and afucosylated glycans remained stationary. The “PR3-AAV” during the induction treatment, showed a relative increase in digalactosylated glycans (79.7%) and bisecting GlcNAc of (43.5%), accompanied by a relative decrease in agalactosylated glycans (45.4%). Conclusion The pilot cases evidenced IgG N-glycome changes during immunosuppressive treatment administered in both AAV subtypes. The increase in the galactosylated glycans during remission achievement could indicate a transition towards anti-inflammatory IgG “phenotype” both in MPO and PR3 AAV. The interpretation might be similar for the increase in bisecting GlcNAc in PR3 AAV, while for afucosylation and sialylation, straightforward assumptions would be challenging. These pilot data justify further “mining” of IgG N-glycome changes in the search for AAV activity biomarker candidates.

N-glycome analysis of individual proteins and tissues is crucial for fundamental and applied biomedical research and medical diagnosis and plays an important role in the evaluation of the quality of biopharmaceutical and biotechnological products. The interest in this research area continues to grow annually, thereby increasing the demand for the high-throughput profiling of human blood plasma N-glycome. In response to this need, we have developed an optimized, simple, and rapid protocol for the N-glycome profiling of human plasma proteins. This protocol encompasses the entire analysis cycle, from plasma isolation to N-glycan spectrum quantification. While the proposed method may have lower efficiency compared to already published high-throughput methods, its adaptability makes it suitable for implementation in virtually any molecular biological laboratory.

SummaryAll four subclasses of immunoglobulin G (IgG) antibodies have glycan structures attached to the protein part of the IgG molecules. Glycans linked to the Fc portion of IgG are found in all IgG antibodies, while about one‐fifth of IgG antibodies in plasma also have glycans attached to the Fab portion of IgG. The IgG3 subclass is characterized by more complex glycosylation compared to other IgG subclasses. In this review, we discuss the significant influence that glycans exert on the structural and functional properties of IgG. We provide a comprehensive overview of how the composition of these glycans can affect IgG's effector functions by modulating its interactions with Fcγ receptors and other molecules such as the C1q component of complement, which in turn influence various immune responses triggered by IgG, including antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC). In addition, the importance of glycans for the efficacy of therapeutics like monoclonal antibodies and intravenous immunoglobulin (IVIg) therapy is discussed. Moreover, we offer insights into IgG glycosylation characteristics and roles derived from general population, disease‐specific, and interventional studies. These studies indicate that IgG glycans are important biomarkers and functional effectors in health and disease.

Mastitis represents the biggest threat to the health and productivity of dairy cows, leading to substantial economic losses in milk production. It manifests in two forms: clinical mastitis, easily diagnosed by visible symptoms, and subclinical mastitis (SCM), which lacks overt clinical signs. SCM's elusive nature often results in it going undetected, thus facilitating the spread of the disease-causing agent due to lack of treatment. Finding a reliable biomarker for early SCM would reduce the possibility of mastitis spreading in the herd, reduce the need for antibiotic use and ultimately reduce milk losses for producers. Utilizing state-of-the-art proteomics techniques, 138 milk samples from dairy cows in continental Croatia underwent analysis. These samples were categorized into four groups based on the Zagreb Mastitis Test (ZMT) and microbiological analysis: lowSCC- (n = 20), lowSCC + (n = 20), medSCC + (n = 79), and highSCC + (n = 19). A total of 386 proteins were identified and quantified, with 76 proteins showing significant differential abundances among the groups. Many of these proteins are linked to the innate immune system, as well as neutrophil and platelet degranulation processes. Through fold changes observed between groups, 15 proteins exhibiting biomarker characteristics for subclinical mastitis (SCM) were identified. Among these, five proteins-cathelicidins (-1, -4, and -7), lactoferrin, and haptoglobin-showed particular promise.

ABSTRACTWith aging, there is a correlation between a decline in the body’s ability to maintain regular functioning and greater susceptibility to age-related diseases. Therapeutic interventions targeting the underlying biological changes of aging hold promise for preventing or delaying multiple age-related diseases. Metformin, a drug commonly used for diabetes treatment, has emerged as a potential gerotherapeutic agent due to its established safety record and preclinical and clinical data on its anti-aging effects. Glycosylation, one of the most common and complex co- and post-translational protein modifications, plays a crucial role in regulating protein function and has been linked to aging and various diseases. Changes in IgG glycosylation patterns have been observed with age, and these alterations may serve as valuable biomarkers for disease predisposition, diagnosis, treatment monitoring, and overall health assessment. In this study, we analyzed the IgG glycosylation patterns of individuals under treatment with metformin, testosterone, metformin plus testosterone and placebo, and investigated the longitudinal changes in glycosylation over time. We observed statistically significant differences in the IgG glycome composition between participants on testosterone therapy and placebo, with decreased agalactosylation and increased galactosylation and sialylation. However, metformin therapy did not result in statistically significant changes in glycosylation patterns. These findings contribute to our understanding of the impact of therapeutic interventions on IgG glycosylation and confirm the value of IgG glycosylation as a significant biomarker, capable of assessing biological age using the GlycanAge index and providing insight into overall health compared to chronological age.