How Apicomplexa Parasites Secrete and Build Their Invasion Machinery.

Apicomplexa are obligatory intracellular parasites that sense and actively invade host cells. Invasion is a conserved process that relies on the timely and spatially controlled exocytosis of unique specialized secretory organelles termed micronemes and rhoptries. Microneme exocytosis starts first and likely controls the intricate mechanism of rhoptry secretion. To assemble the invasion machinery, micronemal proteins-associated with the surface of the parasite-interact and form complexes with rhoptry proteins, which in turn are targeted into the host cell. This review covers the molecular advances regarding microneme and rhoptry exocytosis and focuses on how the proteins discharged from these two compartments work in synergy to drive a successful invasion event. Particular emphasis is given to the structure and molecular components of the rhoptry secretion apparatus, and to the current conceptual framework of rhoptry exocytosis that may constitute an unconventional eukaryotic secretory machinery closely related to the one described in ciliates.

An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

A Plasmodium falciparum C-mannosyltransferase is dispensable for parasite asexual blood stage development.

C-mannosylation was recently identified in the thrombospondin-related anonymous protein (TRAP) from Plasmodium falciparum salivary gland sporozoites. A candidate P. falciparum C-mannosyltransferase (PfDPY-19) was demonstrated to modify thrombospondin type 1 repeat (TSR) domains in vitro, exhibiting a different acceptor specificity than their mammalian counterparts. According to the described minimal acceptor of PfDPY19, several TSR domain-containing proteins of P. falciparum could be C-mannosylated in vivo. However, the relevance of this protein modification for the parasite viability remains unknown. In the present study, we used CRISPR/Cas9 technology to generate a PfDPY19 null mutant, demonstrating that this glycosyltransferase is not essential for the asexual blood development of the parasite. PfDPY19 gene disruption was not associated with a growth phenotype, not even under endoplasmic reticulum-stressing conditions that could impair protein folding. The data presented in this work strongly suggest that PfDPY19 is unlikely to play a critical role in the asexual blood stages of the parasite, at least under in vitro conditions.

Pre-operative Color Doppler Ultrasonography Predicts Endovenous Heat Induced Thrombosis after Endovenous Radiofrequency Ablation.

OBJECTIVES: The aim was to identify pre-operative color Doppler ultrasound (CDUS) variables predictive of post-operative endovenous heat induced thrombosis (EHIT) after radiofrequency ablation (RFA) of the saphenous veins. DESIGN: This was a single centre, observational study with retrospective analysis of consecutive patients treated from December 2010 to February 2017. MATERIALS AND METHODS: Pre-operatively, the diameter of the sapheno-femoral junction (dSFJ), distance between superficial epigastric vein and SFJ (dSEV-SFJ) [corrected], maximum great saphenous vein (GSV) diameter (mdGSV), diameter of the saphenous-popliteal junction (dSPJ), and mean small saphenous vein (SSV) diameter (adSSV) were measured. All patients received low molecular weight heparin (LWMH) at a prophylactic dose for a week. Post-operatively, CDUS was performed after 72 h, 1 week, and 3 months. RESULTS: Venous interventions on 512 patients were performed: 449 (87.7%) underwent RFA of the GSV (Group 1), and 63 (12.3%) of the SSV (Group 2). At Day 3 post-operatively, CDUS documented 100% complete closure of the treated saphenous vein segment. Overall, 40 (7.8%) cases of post-operative EHIT were identified: 29 in Group 1, and 11 in Group 2 (6.4% vs. 17.5%, p = .005). Deep venous thrombosis or pulmonary embolism did not occur in either group. At the 1 month follow up, all cases of EHIT regressed. In Group 1, on multivariate analysis, dSEV-SFJ [corrected] (OR, 1.13, p = .036; 95% CI 1.01-1.27) was the only statistically significant predictor for EHIT. A dSEV-SFJ [corrected] distance of 4.5 mm yielded an 84% of sensitivity for EHIT prediction with a 72.4% positive predictive value. In Group 2, univariate analysis did not identify independent risk factors for EHIT occurrence. CONCLUSIONS: EHIT was higher than previously reported. The dSEV-SFJ [corrected] was the most significant predictor for EHIT in the GSV group. A greater distance between the tip of the radiofrequency catheter and the SFJ may decrease the risk of developing this complication.

Activities at Thoracic Aortic Research Center, IRCCS Policlinico San Donato.

The Thoracic Aortic Research Center (TARC) of the IRCCS Policlinico San Donato (PSD) aims to promote research on thoracic aortic diseases, to disclose the scientific knowledge and clinical experience and to develop new scientific paths within the Hospital and the aortic community, in collaboration with other national and international centres. Thoracic Aortic Research Center collaborates with many centres in both Europe (e.g. University of Utrecht, the Netherlands) and the USA (e.g. University of Michigan). This has led to multiple highly regarded publications in respected cardiovascular journals and has led to several PhD programmes resulting in doctorate degrees. Within Italy, in association with the Bioengineering School of the University of Pavia, TARC has founded the "BETA-lab" (Biomechanics for Endovascular Treatment of the Aorta laboratory), where MDs, Bioengineers, and PhD fellows conduct experimental studies using in vitro/ex vivo models of the physiologic aorta and aortic diseases. Furthermore, a database (iCardiocloud) where the medical imaging of cardiovascular patients from the PSD is structured, for in silico analysis utilizing computational fluid dynamics, and in vitro studies using also 3D printed aortic models. With the role of principal investigator or co-investigator, TARC at PSD has been participating in other several projects, including the International Registry of Acute Aortic Dissection, the International Aortic Arch Surgery Study Group, the European Registry of Endovascular Aortic Repair Complications, the ADSORB and ASSIST trials, and the GREAT registry. International collaborations have included also studies on predictors of aortic growth after dissection with the Yale University and University of Virginia, and on aortic biomarkers with the University of Tokyo.

Cross-species comparison of mammalian saliva using an LC-MALDI based proteomic approach.

Despite the importance of saliva in the regulation of oral cavity homeostasis, few studies have been conducted to quantitatively compare the saliva of different mammal species. Aiming to define a proteome signature of mammals' saliva, an in-depth SDS-PAGE-LC coupled to MS/MS (GeLC-MS/MS) approach was used to characterize the saliva from primates (human), carnivores (dog), glires (rat and rabbit), and ungulates (sheep, cattle, horse). Despite the high variability in the number of distinct proteins identified per species, most protein families were shared by the mammals studied with the exception of cattle and horse. Alpha-amylase is an example that seems to reflect the natural selection related to digestion efficacy and food recognition. Casein protein family was identified in all species but human, suggesting an alternative to statherin in the protection of hard tissues. Overall, data suggest that different proteins might assure a similar role in the regulation of oral cavity homeostasis, potentially explaining the specific mammals' salivary proteome signature. Moreover, some protein families were identified for the first time in the saliva of some species, the presence of proline-rich proteins in rabbit's saliva being a good example.

Sugar activation and glycosylation in Plasmodium.

Glycoconjugates are important mediators of host-pathogen interactions and are usually very abundant in the surface of many protozoan parasites. However, in the particular case of Plasmodium species, previous works show that glycosylphosphatidylinositol anchor modifications, and to an unknown extent, a severely truncated N-glycosylation are the only glycosylation processes taking place in the parasite. Nevertheless, a detailed analysis of the parasite genome and the recent identification of the sugar nucleotide precursors biosynthesized by Plasmodium falciparum support a picture in which several overlooked, albeit not very prominent glycosylations may be occurring during the parasite life cycle. In this work, the authors review recent developments in the characterization of the biosynthesis of glycosylation precursors in the parasite, focusing on the outline of the possible fates of these precursors.

Antimicrobial peptides: an alternative for innovative medicines?

Antimicrobial peptides are small molecules with activity against bacteria, yeasts, fungi, viruses, bacteria, and even tumor cells that make these molecules attractive as therapeutic agents. Due to the alarming increase of antimicrobial resistance, interest in alternative antimicrobial agents has led to the exploitation of antimicrobial peptides, both synthetic and from natural sources. Thus, many peptide-based drugs are currently commercially available for the treatment of numerous ailments, such as hepatitis C, myeloma, skin infections, and diabetes. Initial barriers are being increasingly overcome with the development of cost-effective, more stable peptides. Herein, we review the available strategies for their synthesis, bioinformatics tools for the rational design of antimicrobial peptides with enhanced therapeutic indices, hurdles and shortcomings limiting the large-scale production of AMPs, as well as the challenges that the pharmaceutical industry faces on their use as therapeutic agents.

Postimplantation syndrome after endovascular aortic repair using the Anaconda™ endograft.

BACKGROUND: We report our retrospective experience on postimplantation syndrome (PIS) after the use of Anaconda™ endograft in patients undergoing elective endovascular repair of abdominal aortic aneurysms (EVAR). METHODS: Data of all patients undergoing elective EVAR between May 2000 and June 2013 using the Anaconda endograft were retrospectively reviewed and the outcomes were analyzed. Preoperative and intraoperative data, any early postoperative complications, length of in-hospital stay, incidence of PIS, and long-term complications were recorded in a database. Patients' quality of life (QOL) was also assessed at 1 month after the procedure. Statistical analysis was performed and P values ≤0.05 were considered statistically significant. Chi-squared tests, log-rank tests, Wilcoxon tests, and Kaplan-Meier survival analysis were performed as appropriate. RESULTS: Between May 2000 and June 2013, 118 patients (8 female, 6.7%) underwent elective EVAR using the Anaconda endograft. Primary success was obtained in 117 cases (99.1%). Patients were divided into 2 groups based on either the occurrence of PIS (group A: 24 patients, 20.3%) or not (group B). The length of the procedure and the in-hospital stay were longer for group A. One patient from group B died on second postoperative day from myocardial infarction (0.8%). Mean follow-up was 48.4 months (range 5-162 months). Seven endoleaks occurred in the long term regardless of the development of PIS. We did not find any correlation between the presence of PIS and the occurrence of long-term complications, but PIS was correlated to the preoperative burden of thrombus of the aneurysmal sac. On the other side, analysis of QOL surveys showed that patients who had PIS after surgery felt significantly more limited in carrying out their daily physical activities and were more emotionally discouraged and depressed/anxious about their state of health than the group that did not have PIS. CONCLUSIONS: In our experience, the occurrence of PIS was related to the duration of the procedure and the preoperative burden of thrombus of the aneurysmal sac. Overall, PIS was a benign complication after EVAR using the Anaconda endograft. However, it affected significantly the length of the in-hospital stay. Moreover, patients who had PIS after surgery felt significantly more limited in carrying out their daily physical activities and were more emotionally discouraged and depressed/anxious about their state of health than the group that did not have PIS.

Effects of positive airway pressure on basilic vein diameter and venous flow velocity in healthy volunteers.

INTRODUCTION: The placement of vascular catheters of adequate size in accordance to catheter-to-vein ratio (CVR) recommendations represents one of the cornerstones of catheter-related upper vein thrombosis prevention. However there is scarcity of data on its effect on the venous dynamics of the basilic vein, a common site for long-term catheter placement. This study investigates the effects of the application of positive airway pressure on the diameter and blood flow velocity of basilic vein. We also measured the effects of under-armpit straps, a device commonly used to keep continuous positive airway pressure (CPAP) helmets in place. METHODS: We enrolled 28 healthy volunteers. Basilic vein diameter and minimum/maximum blood flow velocity, according to respiratory venous flow oscillation, were measured by ultrasound on the midpoint of their dominant arm during spontaneous breathing and during breathing in a CPAP helmet with 10 cm H2O of airway pressure applied, with the helmet kept in place either through armpit straps or by tying the helmet to the bed. RESULTS: The application of 10 cm H2O of positive airway pressure significantly increased basilic vein diameter by 0.9 ± 0.2 mm, while reducing minimum blood flow velocity by 1.8 ± 0.4 cm/s. These effects were amplified by the application of under armpit straps. CONCLUSIONS: Breathing with positive airway pressure increases basilic vein diameter while reducing blood flow-velocity. This phenomenon might lead to an incorrect assessment of CVR, misleading the operator into choosing improperly large catheters.

Rhoptry secretion system structure and priming in Plasmodium falciparum revealed using in situ cryo-electron tomography.

Apicomplexan parasites secrete contents of the rhoptries, club-shaped organelles in the apical region, into host cells to permit their invasion and establishment of infection. The rhoptry secretory apparatus (RSA), which is critical for rhoptry secretion, was recently discovered in Toxoplasma and Cryptosporidium. It is unknown whether a similar molecular machinery exists in the malaria parasite Plasmodium. In this study, we use in situ cryo-electron tomography to investigate the rhoptry secretion system in P. falciparum merozoites. We identify the presence of an RSA at the cell apex and a morphologically distinct apical vesicle docking the tips of the two rhoptries to the RSA. We also discover two additional rhoptry organizations that lack the apical vesicle. Using subtomogram averaging, we reveal different conformations of the RSA structure corresponding to different rhoptry organizations. Our results highlight previously unknown steps in the process of rhoptry secretion and indicate a regulatory role for the conserved apical vesicle in host invasion by apicomplexan parasites.

An Alveolata secretory machinery adapted to parasite host cell invasion.

Apicomplexa are unicellular eukaryotes and obligate intracellular parasites, including Plasmodium (the causative agent of malaria) and Toxoplasma (one of the most widespread zoonotic pathogens). Rhoptries, one of their specialized secretory organelles, undergo regulated exocytosis during invasion1. Rhoptry proteins are injected directly into the host cell to support invasion and subversion of host immune function2. The mechanism by which they are discharged is unclear and appears distinct from those in bacteria, yeast, animals and plants. Here, we show that rhoptry secretion in Apicomplexa shares structural and genetic elements with the exocytic machinery of ciliates, their free-living relatives. Rhoptry exocytosis depends on intramembranous particles in the shape of a rosette embedded into the plasma membrane of the parasite apex. Formation of this rosette requires multiple non-discharge (Nd) proteins conserved and restricted to Ciliata, Dinoflagellata and Apicomplexa that together constitute the superphylum Alveolata. We identified Nd6 at the site of exocytosis in association with an apical vesicle. Sandwiched between the rosette and the tip of the rhoptry, this vesicle appears as a central element of the rhoptry secretion machine. Our results describe a conserved secretion system that was adapted to provide defence for free-living unicellular eukaryotes and host cell injection in intracellular parasites.

Reduced Placental Transfer of Antibodies Against a Wide Range of Microbial and Vaccine Antigens in HIV-Infected Women in Mozambique.

Transplacental transfer of antibodies is essential for conferring protection in newborns against infectious diseases. We assessed the impact of different factors, including gestational age and maternal infections such as HIV and malaria, on the efficiency of cord blood levels and placental transfer of IgG subclasses. We measured total IgG and IgG subclasses by quantitative suspension array technology against 14 pathogens and vaccine antigens, including targets of maternal immunization, in 341 delivering HIV-uninfected and HIV-infected mother-infant pairs from southern Mozambique. We analyzed the association of maternal HIV infection, Plasmodium falciparum exposure, maternal variables and pregnancy outcomes on cord antibody levels and transplacental transfer. Our results show that maternal antibody levels were the main determinant of cord antibody levels. Univariable and multivariable analysis showed that HIV reduced the placental transfer and cord levels of IgG and IgG1 principally, but also IgG2 to half of the antigens tested. P. falciparum exposure and prematurity were negatively associated with cord antibody levels and placental transfer, but this was antigen-subclass dependent. Our findings suggest that lower maternally transferred antibodies may underlie increased susceptibility to infections of HIV-exposed infants. This could affect efficacy of maternal vaccination, especially in sub-Saharan Africa, where there is a high prevalence of HIV, malaria and unfavorable environmental factors.

Laparoscopic Clipping of the Inferior Mesenteric Artery and Intraoperative Indocyanine Green Angiography for Type II Endoleak Following Endovascular Aneurysm Repair.

Background: Type II endoleaks from a patent inferior mesenteric artery (IMA) occur in up to one-third of patients undergoing endovascular repair of abdominal aortic aneurysms. In the majority of patients, retrograde flow in the aneurysmal sac outside the endograft will seal over time and is rarely associated with sac enlargement or aortic rupture. Intervention is generally recommended in patients with progressively enlarging endoleaks, especially when the sac diameter increases >10 mm during the follow-up, and endovascular IMA embolization has a high rate of treatment failure. Methods: We report a procedure of laparoscopic IMA clipping combined with intraoperative indocyanine green (ICG) angiography to confirm vascular anatomy, colonic perfusion, and the technical success of the procedure. Results: Three selected octogenarian patients with persistent type II endoleak after endovascular repair of abdominal aortic aneurysm underwent IMA clipping with ICG angiography. Mean operative time was 58 ± 9 minutes. There were no procedure-related complications, and no hypersensitivity reactions nor other side effects associated with ICG dye administration occurred. All patients were discharged home on postoperative day 1 and are asymptomatic and free of recurrence at a mean follow-up of 15 months. Conclusions: Laparoscopic IMA clipping is a safe remedial procedure in patients with type II endoleak after endovascular repair of abdominal aortic aneurysms.

Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate N-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development.

UDP-N-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and N- and O-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate N-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual Plasmodium falciparum parasites. Furthermore, we present the 1.95 Å resolution structure of the GNA1 ortholog from Cryptosporidium parvum, an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for P. falciparum GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria.IMPORTANCE Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite's ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate N-acetyltransferase (GNA1), required for the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), is essential for P. falciparum asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite C. parvum, used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target.

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.

Toward Neuroproteomics in Biological Psychiatry: A Systems Approach Unravels Okadaic Acid-Induced Alterations in the Neuronal Phosphoproteome.

Neuroproteomics is an evolving field of postgenomic medicine, highlighting the convergence of psychiatry/neurology and proteomics, yet compared with neurogenetics, it has received little attention. This study in rat primary neuronal cultures provides an example of a neuroproteomic approach relevant to the study of psychiatric disease pathophysiology, focusing on Alzheimer's disease. In this context, okadaic acid (OA) is routinely used in experimental designs to investigate phosphorylation-mediated events. It is a potent protein phosphatase (PP) inhibitor, particularly of PP1 and PP2A. Typically, a single protein and its phosphorylation level are monitored upon OA exposure. Although useful, this can be misleading as protein phosphorylation-mediated events involve complex signaling cascades and an array of kinases, phosphatases, and substrates. Bearing in mind the involvement of multiple pathways and cascade cross talk, this study employed a systems approach to analyze OA-induced molecular responses through PP inhibition. We showed that upon OA exposure, the recovery rate of 245 phosphoproteins significantly increased, while that of 75 significantly decreased. The prominent biological processes affected included anatomical structural development, transport, cell differentiation, and signal transduction. The associated phosphointeraction networks identified nodes representing OA-responsive phosphoproteins. Many of these are key players of signaling cascades relevant to a range of pathologies. In summary, the data presented results from a neuroproteomic preclinical study offering an array of phosphoproteins as potential targets for future diagnostic and therapeutic strategies in biological psychiatry. We note, however, the nonspecificity of targeting PPs themselves and emphasize the need for future neuroproteomic approaches toward systems psychiatry.

Altered protein phosphorylation as a resource for potential AD biomarkers.

The amyloidogenic peptide, Aß, provokes a series of events affecting distinct cellular pathways regulated by protein phosphorylation. Aß inhibits protein phosphatases in a dose-dependent manner, thus it is expected that the phosphorylation state of specific proteins would be altered in response to Aß. In fact several Alzheimer's disease related proteins, such as APP and TAU, exhibit pathology associated hyperphosphorylated states. A systems biology approach was adopted and the phosphoproteome, of primary cortical neuronal cells exposed to Aß, was evaluated. Phosphorylated proteins were recovered and those whose recovery increased or decreased, upon Aß exposure across experimental sets, were identified. Significant differences were evident for 141 proteins and investigation of their interactors revealed key protein clusters responsive to Aß treatment. Of these, 73 phosphorylated proteins increased and 68 decreased upon Aß addition. These phosphorylated proteins represent an important resource of potential AD phospho biomarkers that should be further pursued.

Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins.

Protein post-translational modifications (PTMs) have increasingly become a research field of incredible importance to fully understand the regulation of biological processes in health and disease. Among PTMs, glycosylation is one of the most studied for which contributed the development and improvement of enrichment techniques. Nowadays, glycoprotein enrichment methods are based on lectin affinity, covalent interactions, and hydrophilic interaction liquid chromatography (HILIC). Nonetheless, the nanotechnology era has fetched new methods to enrich glycoproteins from complex samples as human biological fluids. For instance, magnetic nanoparticles (MNPs) are being used as an interesting enrichment approach allowing a better characterization of glycoproteins and glycopeptides.In this chapter, we describe an enrichment method based on MNPs functionalized with lectins (Concavalin A, wheat germ agglutinin, and Maackia amurensis lectin) to enrich specific sets of glycoproteins from biological fluids. Moreover, it is proposed a bioinformatic strategy to deal with data retrieved from mass spectrometry analysis of enriched samples aiming the identification of relevant biological processes modulated by a given stimuli and, ultimately, of new biomarkers for disease screening/management.