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Breast cancer brain metastasis has been recognized as one of the central issues in breast cancer research. The elucidation of the processes and pathways that mediate this step will provide important clues for a better understanding of breast cancer metastasis. Increasing evidence suggests that aberrant glycosylation patterns greatly contribute to cell invasion and cancer metastasis. Herein, we combined next-generation RNA sequencing with liquid chromatography-tandem mass spectrometry-based proteomic and N-glycomic analysis from five breast cancer cell lines and one brain cancer cell line to investigate the possible mechanisms of breast cancer brain metastasis. The genes/proteins associated with cell movement were highlighted in breast cancer brain metastasis. The integrin signaling pathway and the up-regulation of α-integrin (ITGA2, ITGA3) were associated with the brain metastatic process. 12 glycogenes showed unique expression in 231BR, which could result in an increase of sialylation during brain metastasis. In agreement with the changes of glycogenes, 60 out of 63 N-glycans that were identified exhibited differential expression among cell lines. The correlation between glycogenes and glycans revealed the importance of sialylation and sialylated glycans in breast cancer brain metastasis. Highly sialylated N-glycans, which were up-regulated in brain-seeking cell line 231BR, likely play a role in brain metastasis.

Neurofibromatosis type 1 (NF1) is associated with social dysfunction in children. Here the authors show that Nf1 heterozygous mice have deficits in social memory associated with alterations in amygdala plasticity and Map kinase signaling. Global deletion or amygdala-specific pharmacological inhibition of Pak1 rescued social deficits in Nf1 heterozygous mice. Children with neurofibromatosis type 1 (NF1) are increasingly recognized as having a high prevalence of social difficulties and autism spectrum disorders (ASDs). We demonstrated a selective social learning deficit in mice with deletion of a single Nf1 allele ( Nf1 +/− ), along with greater activation of the mitogen-activated protein kinase pathway in neurons from the amygdala and frontal cortex, structures that are relevant to social behaviors. The Nf1 +/− mice showed aberrant amygdala glutamate and GABA neurotransmission, deficits in long-term potentiation and specific disruptions in the expression of two proteins that are associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (Adam22) and heat shock protein 70 (Hsp70), respectively. All of these amygdala disruptions were normalized by the additional deletion of the p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1 +/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide insights and therapeutic targets for patients with NF1 and ASDs.

The success of symbioses between cnidarian hosts (e.g., corals and sea anemones) and micro-algal symbionts hinges on the molecular interactions that govern the establishment and maintenance of intracellular mutualisms. As a fundamental component of innate immunity, glycan-lectin interactions impact the onset of marine endosymbioses, but our understanding of the effects of cell surface glycome composition on symbiosis establishment remains limited. In this study, we examined the canonical N-glycan biosynthesis pathway in the genome of the dinoflagellate symbiont Breviolum minutum (family Symbiodiniaceae) and found it to be conserved with the exception of the transferase GlcNAc-TII (MGAT2). Using coupled liquid chromatography-mass spectrometry (LC-MS/MS), we characterized the cell surface N-glycan content of B. minutum, providing the first insight into the molecular composition of surface glycans in dinoflagellates. We then used the biosynthesis inhibitors kifunensine and swainsonine to alter the glycan composition of B. minutum. Successful high-mannose enrichment via kifunensine treatment resulted in a significant decrease in colonization of the model sea anemone Aiptasia (Exaiptasia pallida) by B. minutum. Hybrid glycan enrichment via swainsonine treatment, however, could not be confirmed and did not impact colonization. We conclude that functional Golgi processing of N-glycans is critical for maintaining appropriate cell surface glycan composition and for ensuring colonization success by B. minutum.

T-cell malignancies represent a group of complex cancers arising from T cells and include aggressive subtypes such as Adult T-cell Leukemia/Lymphoma (ATL) and T-cell Acute Lymphoblastic Leukemia (T-ALL). Patients with these aggressive subtypes still represent an unmet medical condition. The synthetic adamantyl retinoid ST1926, a potent DNA polymerase-α inhibitor, proved a promising potency in preclinical models of ATL and peripheral T-cell lymphoma. Using advanced liquid chromatography–mass spectrometry (LC–MS/MS) techniques, we explored the effects of ST1926 on global protein expression in ATL (HuT-102) and T-ALL (MOLT-4) cells. We demonstrate that ST1926 triggers differentiation and apoptosis in malignant T-cells while halting tumor progression. Evidence at the proteomics level reveals the impact of ST1926 on crucial DNA replication enzymes and cell cycle regulation, highlighting its potential to reduce leukemogenesis and promote apoptosis. Our findings underscore the potential of ST1926 as an innovative therapeutic approach to address these aggressive T-cell malignancies, providing valuable insights into developing new targeted therapies and improving the outcomes and prognosis of patients with these challenging diseases.

Cerebrospinal fluid (CSF) contains valuable biological and neurological information. However, its glycomics analysis is hampered due to the low amount of protein in the biofluid, as has been demonstrated by other glycomics studies using a substantial amount of CSF. In this work, we investigated different N-glycan sample preparation approaches to develop a more sensitive method. These methods, one with an increased amount of buffer solution during the N-glycan release step with a lower amount of sample volume and the other with Filter-Aided N-Glycan Separation (FANGS), were compared with recent work to demonstrate their effectiveness. It was demonstrated that an increased amount of buffer solution showed higher intensity in comparison to the previously published method and FANGS. This suggested that digestion efficiency during the N-glycan release step was not in an optimal condition from the previously published method, and that there is a substantial loss of sample with FANGS when preparing N-glycans from CSF.

Small extracellular vesicles (sEVs) are gaining recognition as potential biomarkers for diseases, including cancer, due to their involvement in key pathophysiological processes. However, the glycosylation of EVs and the specific roles of their glycans remain poorly understood. While several methods exist for isolating sEVs from complex biological samples, achieving sufficient purity and quantity for mass spectrometry-based glycoproteomic analysis remains a significant challenge. In this study, we compared two commonly used isolation methods, ultracentrifugation (UC) and immunoaffinity capture (MagCapture kit), across different starting volumes of human serum (200 µL and 500 µL) to evaluate their performance for downstream glycoproteomic analysis. While prior studies have examined protein content across isolation methods, our work uniquely investigates how isolation technique and sample volume affect glycoproteomic yield and quality. We show that UC, particularly at higher sample volumes, enables deeper glycoproteomic coverage, whereas MagCapture is advantageous when serum availability is limited. Notably, we report for the first time site-specific glycan microheterogeneity on sEV glycoproteins derived from human serum, including multiple glycoforms at the same glycosylation site. These findings highlight the complexity and biological relevance of glycosylation in sEV proteins and offer practical guidance for optimizing isolation protocols based on specific omics applications.

Salmonella enterica serovar Typhimurium ( S . Typhimurium) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, S . Typhimurium produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species, and the S . Typhimurium genome contains two annotated chitinases: STM0018 ( chiA ) and STM0233 . However, the role of these chitinases during S . Typhimurium pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously, and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. S . Typhimurium lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro . In a gastroenteritis mouse model, chitinase-deficient S . Typhimurium strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed S . Typhimurium dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type S . Typhimurium, suggesting that chitinases are secreted. By analyzing N -linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of S . Typhimurium chitinases. This analysis also revealed a differential abundance of Lewis X/A-containing glycans that is likely a result of host cell modulation due to the detection of S . Typhimurium chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that S . Typhimurium chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome.

Glycosylation is one of the most common and complex post-translational modifications of proteins. However, there are other carbohydrates such as free oligosaccharides and glycosphingolipids-glycans that are associated with important biological and clinical roles. To analyze these molecules using liquid chromatography coupled with mass spectrometry (LC-MS), the permethylation approach was utilized. Although permethylation is a commonly utilized glycan derivatization technique, separation of permethylated glycans released from glycosphingolipid (GSL) by LC-MS has never been previously demonstrated. Here, a nanoflow porous graphitized carbon (PGC) column coupled with a high-resolution mass spectrometer was used to achieve isomeric separation of these permethylated glycans. We demonstrate the separation of free reducing end and reduced end O-glycans, free oligosaccharides derived from human milk, and GSL glycans derived from the MDA-MB-231BR cancer cell line using PGC-LC-MS.

Glycosylation, the enzymatic addition of glycans to proteins and lipids, is a critical post-translational modification that influences protein folding, stability, trafficking, immune modulation, and cell signaling. The vast structural diversity of glycans arising from differences in monosaccharide composition, branching, and terminal modifications such as sialylation, fucosylation, and sulfation underpins their functional specificity and regulatory capacity. This review provides a comprehensive overview of glycan biosynthesis, with a focus on N-glycans, O-glycans, glycosaminoglycans (GAGs), and glycolipids. It explores their essential roles in maintaining cellular homeostasis, development, and immune surveillance. In health, glycans mediate cell–cell communication, protein interactions, and immune responses. In disease, however, aberrant glycosylation is increasingly recognized as a hallmark of numerous pathological conditions, including cancer, neurodegenerative disorders, autoimmune diseases, and a wide range of infectious diseases. Glycomic alterations contribute to tumor progression, immune evasion, therapy resistance, neuroinflammation, and synaptic dysfunction. Tumor-associated carbohydrate antigens (TACAs) and disease-specific glycoforms present novel opportunities for biomarker discovery and therapeutic targeting. Moreover, glycan-mediated host–pathogen interactions are central to microbial adhesion, immune escape, and virulence. This review highlights current advances in glycomics technologies, including mass spectrometry, lectin microarrays, and glycoengineering, which have enabled the high-resolution profiling of the glycome. It also highlights the emerging potential of single-cell glycomics and multi-omics integration in precision medicine. Understanding glycome and its dynamic regulation is essential for uncovering the molecular mechanisms of disease and translating glycomic insights into innovative diagnostic and therapeutic strategies.

Clinical studies have linked glyphosate exposure to substantial morbidity, with acute kidney injury occurring in some cases. Although the toxic effects of glyphosate-based herbicides (GBHs) have been reported in several studies, their molecular impact on renal function remains poorly understood. Given the kidney’s critical role in excretion, it is particularly susceptible to damage from xenobiotic exposure. In this study, we aim to identify N-glycomics and proteomics change in the kidney following chronic GBH exposure, to better understand the mechanisms behind glyphosate-induced kidney damage. Kidney tissues from female and male rats were analyzed using liquid chromatography–tandem mass spectrometry. The results revealed notable changes in the N-glycan composition, particularly in the fucosylated and sialofucosylated N-glycan types. The proteomic analysis revealed the activation of immune signaling and inflammatory pathways, including neutrophil degranulation, integrin signaling, and MHC class I antigen presentation. Transcription regulators, such as IL-6, STAT3, and NFE2L2, were upregulated, indicating a coordinated inflammatory and oxidative stress response. Sex-specific differences were apparent, with female rats exhibiting more pronounced alterations in both the N-glycan and protein expression profiles, suggesting a higher susceptibility to GBH-induced nephrotoxicity. These findings provide new evidence that chronic GBH exposure may trigger immune activation, inflammation, and potentially carcinogenic processes in the kidney.