Micromotion Derived Fluid Shear Stress Mediates Peri-Electrode Gliosis through Mechanosensitive Ion Channels.

The development of bioelectronic neural implant technologies has advanced significantly over the past 5 years, particularly in brain-machine interfaces and electronic medicine. However, neuroelectrode-based therapies require invasive neurosurgery and can subject neural tissues to micromotion-induced mechanical shear, leading to chronic inflammation, the formation of a peri-electrode void and the deposition of reactive glial scar tissue. These structures act as physical barriers, hindering electrical signal propagation and reducing neural implant functionality. Although well documented, the mechanisms behind the initiation and progression of these processes are poorly understood. Herein, in silico analysis of micromotion-induced peri-electrode void progression and gliosis is described. Subsequently, ventral mesencephalic cells exposed to milliscale fluid shear stress in vitro exhibited increased expression of gliosis-associated proteins and overexpression of mechanosensitive ion channels PIEZO1 (piezo-type mechanosensitive ion channel component 1) and TRPA1 (transient receptor potential ankyrin 1), effects further confirmed in vivo in a rat model of peri-electrode gliosis. Furthermore, in vitro analysis indicates that chemical inhibition/activation of PIEZO1 affects fluid shear stress mediated astrocyte reactivity in a mitochondrial-dependent manner. Together, the results suggest that mechanosensitive ion channels play a major role in the development of a peri-electrode void and micromotion-induced glial scarring at the peri-electrode region.

A new multiplex SARS-CoV-2 antigen microarray showed correlation of IgG, IgA, and IgM antibodies from patients with COVID-19 disease severity and maintenance of relative IgA and IgM antigen binding over time.

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity different from the same isotype in whole serum, probably due to competition from the other isotypes present. Using purified antibody isotypes from naïve Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with binding to the S region S1 expressed in insect cells (S1 Sf21) significant for IgG, IgA, and IgM. Assessing longitudinal response for constant concentrations of purified antibody isotypes for a patient subset demonstrated that the relative proportion of antigen-specific IgGs decreased over time for severe disease, but the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen-specific serum IgA and IgM playing a role in maintaining longer-term protection, important for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions.

Lectin microarray profiling demonstrates equivalent global glycosylation for whey protein ingredients enriched with α-lactalbumin and milk fat globule membrane.

Human milk fat globule membrane (MFGM) and whey proteins are nutritionally and functionally valuable, with many beneficial bioactivities associated with their glycosylation. However glycosylation of milk components other than free milk oligosaccharides are underinvestigated. Whey protein concentrate (WPC) ingredients with various enrichments or depletions are used in infant formula (IF) formulations to contribute to human milk equivalence and bioactivity benefits, but their overall or global glycosylation has not been compared. We compared the global glycosylation of commercial WPC ingredients for use in various IF formulations; two MFGM-enriched WPC ingredients (high fat HF1 and lower fat HF2), an α-lactalbumin-enriched WPC (WPC Lac) which has α-lactalbumin concentration closer to human milk and significantly less ß-lactoglobulin which is not present in human milk, and two base WPC ingredients (WPC 80 and WPC 35) using lectin microarray profiling. WPC Lac and WPC HF1 glycosylation were highly similar to each other and both somewhat similar to WPC 35, while WPC HF2 was more similar to the base WPC 80 ingredient. N-linked glycosylation analysis demonstrated that WPC HF1 and WPC Lac were qualitatively most similar to one another, with WPC 80 and WPC 35 having similar structures, confirming lectin microarray profiling as a valuable method to compare global glycosylation. Thus WPC Lac may be a valuable ingredient for providing equivalent glycosylation to MFGM supplementation.

An insight into new glycotherapeutics in glial inflammation: Understanding the role of glycosylation in mitochondrial function and acute to the chronic phases of inflammation.

INTRODUCTION: Glycosylation plays a critical role during inflammation and glial scar formation upon spinal cord injury (SCI) disease progression. Astrocytes and microglia are involved in this cascade to modulate the inflammation and tissue remodeling from acute to chronic phases. Therefore, understating the glycan changes in these glial cells is paramount. METHOD AND RESULTS: A lectin microarray was undertaken using a cytokine-driven inflammatory mixed glial culture model, revealing considerable differential glycosylation from the acute to the chronic phase in a cytokine-combination generated inflamed MGC model. It was found that several N- and O-linked glycans associated with glia during SCI were differentially regulated. Pearson's correlation hierarchical clustering showed that groups were separated into several clusters, illustrating the heterogenicity among the control, cytokine combination, and LPS treated groups and the day on which treatment was given. Control and LPS treatments were observed to be in dense clusters. This was further confirmed with lectin immunostaining in which GalNAc, GlcNAc, mannose, fucose and sialic acid-binding residues were detected in astrocytes and microglia. However, the sialyltransferase inhibitor inhibited this modification (upregulation of the sialic acid expression), which indeed modulates the mitochondrial functions. CONCLUSIONS: The present study is the first functional investigation of glycosylation modulation in a mixed glial culture model, which elucidates the role of the glycome in neuroinflammation in progression and identified potential therapeutic targets for future glyco therapeutics in neuroinflammation.

Metabolic reprogramming and membrane glycan remodeling as potential drivers of zebrafish heart regeneration.

The ability of the zebrafish heart to regenerate following injury makes it a valuable model to deduce why this capability in mammals is limited to early neonatal stages. Although metabolic reprogramming and glycosylation remodeling have emerged as key aspects in many biological processes, how they may trigger a cardiac regenerative response in zebrafish is still a crucial question. Here, by using an up-to-date panel of transcriptomic, proteomic and glycomic approaches, we identify a metabolic switch from mitochondrial oxidative phosphorylation to glycolysis associated with membrane glycosylation remodeling during heart regeneration. Importantly, we establish the N- and O-linked glycan structural repertoire of the regenerating zebrafish heart, and link alterations in both sialylation and high mannose structures across the phases of regeneration. Our results show that metabolic reprogramming and glycan structural remodeling are potential drivers of tissue regeneration after cardiac injury, providing the biological rationale to develop novel therapeutics to elicit heart regeneration in mammals.

Distinct Glycosylation Responses to Spinal Cord Injury in Regenerative and Nonregenerative Models.

Traumatic spinal cord injury (SCI) results in disruption of tissue integrity and loss of function. We hypothesize that glycosylation has a role in determining the occurrence of regeneration and that biomaterial treatment can influence this glycosylation response. We investigated the glycosylation response to spinal cord transection in Xenopus laevis and rat. Transected rats received an aligned collagen hydrogel. The response compared regenerative success, regenerative failure, and treatment in an established nonregenerative mammalian system. In a healthy rat spinal cord, ultraperformance liquid chromatography (UPLC) N-glycoprofiling identified complex, hybrid, and oligomannose N-glycans. Following rat SCI, complex and outer-arm fucosylated glycans decreased while oligomannose and hybrid structures increased. Sialic acid was associated with microglia/macrophages following SCI. Treatment with aligned collagen hydrogel had a minimal effect on the glycosylation response. In Xenopus, lectin histochemistry revealed increased levels of N-acetyl-glucosamine (GlcNAc) in premetamorphic animals. The addition of GlcNAc is required for processing complex-type glycans and is a necessary foundation for additional branching. A large increase in sialic acid was observed in nonregenerative animals. This work suggests that glycosylation may influence regenerative success. In particular, loss of complex glycans in rat spinal cord may contribute to regeneration failure. Targeting the glycosylation response may be a promising strategy for future therapies.

A cellular and molecular analysis of SoxB-driven neurogenesis in a cnidarian.

Neurogenesis is the generation of neurons from stem cells, a process that is regulated by SoxB transcription factors (TFs) in many animals. Although the roles of these TFs are well understood in bilaterians, how their neural function evolved is unclear. Here, we use Hydractinia symbiolongicarpus, a member of the early-branching phylum Cnidaria, to provide insight into this question. Using a combination of mRNA in situ hybridization, transgenesis, gene knockdown, transcriptomics, and in vivo imaging, we provide a comprehensive molecular and cellular analysis of neurogenesis during embryogenesis, homeostasis, and regeneration in this animal. We show that SoxB genes act sequentially at least in some cases. Stem cells expressing Piwi1 and Soxb1, which have broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis. Hydractinia neural cells differentiate while migrating from the aboral to the oral end of the animal, but it is unclear whether migration per se or exposure to different microenvironments is the main driver of their fate determination. Our data constitute a rich resource for studies aiming at addressing this question, which is at the heart of understanding the origin and development of animal nervous systems.

Neoglycoprotein and Glycoprotein Printing on a Hydrogel Functionalized Microarray Surface and Incubation with Labeled Lectins.

Glycan microarrays are widely used to elucidate carbohydrate binding specificity and affinity of various analytes including proteins, microorganisms, cells, and tissues. Glycan microarrays comprise a wide variety of platforms, differing in surface chemistry, presentation of carbohydrates, carbohydrate valency, and detection strategies, all of which impact on analyte performance. This chapter describes detailed methods for printing neoglycoprotein and glycoprotein microarrays on hydrogel-coated slides and incubation of these glycan microarrays with fluorescently labeled lectins.

Calculating Half Maximal Inhibitory Concentration (IC50) Values from Glycomics Microarray Data Using GraphPad Prism.

Half maximal inhibitory concentration (IC50) is a measurement often used to compare the efficiency of various carbohydrates and their derivatives for inhibition of lectin binding to particular ligands. IC50 values can be calculated using experimental data from various platforms including enzyme-linked immunosorbent assay- (ELISA-)type microtiter plate assays, isothermal titration calorimetry (ITC), or glycan microarrays. In this chapter, we describe methods to fluorescently label a lectin, to carry out a lectin binding inhibition experiment on glycan microarrays, and to calculate the IC50 value of a binding inhibitory molecule using GraphPad Prism software. In the example used to illustrate the method in this chapter, IC50 calculation is demonstrated for inhibition of Maackia amurensis agglutinin (MAA) binding to 3'sialyl-N-acetyllactosamine (3SLN) using free lactose.

Mucin Purification and Printing Natural Mucin Microarrays.

Mucin glycosylation is the key facilitator of microbial attachment and nutrition and it varies according to biological location, health and disease status, microbiome composition, infection, and multiple other factors. Mucin glycans have also been reported to attenuate pathogen virulence and mediate biofilm dispersal. With the labor intensive and time-consuming purification required for natural mucins and their low quantitative yield from biological sources, natural mucin microarrays provide a convenient and multiplexed platform to study mucin glycosylation and interactions. In this chapter we describe the purification of natural mucins, using sputum as an example biological source, and the printing of natural mucin microarrays.

Preparation and Fluorescent Labeling of Cell-Derived Micelles and Profiling on Glycan Microarrays.

Mammalian cell surface lectins mediate many important biological interactions which regulate physiological processes and therefore profiling mammalian cells on glycan microarray is of interest. However, many whole mammalian cells are not compatible with glycomics microarray formats and instead cell-derived micelles are prepared and profiled instead of whole cells as they can accurately represent the parental cell glycome. In this chapter, we describe the preparation of cell-derived micelles from mammalian cells, their labeling using a membrane-incorporating dye, and their profiling on a glycan microarray platform.

Bacterial Staining and Profiling for Glycan Interactions on Glycan Microarrays for t-Test Calculation.

The use of glycan microarrays to study carbohydrate interactions of bacterial cells is of great interest owing to the key roles these interactions play in bacterial colonization and infection of a host. In this chapter, the methods to fluorescently stain Gram-positive or Gram-negative bacteria and profiling them for glycan interactions using glycan microarrays are described in detail. The application of the Student's t-test to glycan microarray data using an example data set comparing glycan microarray binding of an Acinetobacter baumannii wild type and mutant strain is also described in step-by-step detail.

Lectin Histochemistry for Tissues and Cells, and Dual Lectin and Antibody Co-localization.

In this chapter we describe in detail methods for lectin staining of (1) tissues, and (2) cells to identify and map endogenous glycosylation. We also describe (3) dual antibody and lectin staining of tissues to associate glycosylation with particular proteins or cells in tissues.

A Self-Powered Piezo-Bioelectric Device Regulates Tendon Repair-Associated Signaling Pathways through Modulation of Mechanosensitive Ion Channels.

Tendon disease constitutes an unmet clinical need and remains a critical challenge in the field of orthopaedic surgery. Innovative solutions are required to overcome the limitations of current tendon grafting approaches, and bioelectronic therapies show promise in treating musculoskeletal diseases, accelerating functional recovery through the activation of tissue regeneration-specific signaling pathways. Self-powered bioelectronic devices, particularly piezoelectric materials, represent a paradigm shift in biomedicine, negating the need for battery or external powering and complementing existing mechanotherapy to accelerate the repair processes. Here, the dynamic response of tendon cells to a piezoelectric collagen-analogue scaffold comprised of aligned nanoscale fibers made of the ferroelectric material poly(vinylidene fluoride-co-trifluoroethylene) is shown. It is demonstrated that motion-powered electromechanical stimulation of tendon tissue through piezo-bioelectric device results in ion channel modulation in vitro and regulates specific tissue regeneration signaling pathways. Finally, the potential of the piezo-bioelectronic device in modulating the progression of tendinopathy-associated processes in vivo, using a rat Achilles acute injury model is shown. This study indicates that electromechanical stimulation regulates mechanosensitive ion channel sensitivity and promotes tendon-specific over non-tenogenic tissue repair processes.

Correction: Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators.

Correction for 'Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators' by Marie Le Berre et al., Mol. Omics, 2021, 17, 338-346, DOI: 10.1039/D0MO00193G.

Burkholderia cenocepacia BCAM2418-induced antibody inhibits bacterial adhesion, confers protection to infection and enables identification of host glycans as adhesin targets.

Trimeric Autotransporter Adhesins (TAA) found in Gram-negative bacteria play a key role in virulence. This is the case of Burkholderia cepacia complex (Bcc), a group of related bacteria able to cause infections in patients with cystic fibrosis. These bacteria use TAAs, among other virulence factors, to bind to host protein receptors and their carbohydrate ligands. Blocking such contacts is an attractive approach to inhibit Bcc infections. In this study, using an antibody produced against the TAA BCAM2418 from the epidemic strain Burkholderia cenocepacia K56-2, we were able to uncover its roles as an adhesin and the type of host glycan structures that serve as recognition targets. The neutralisation of BCAM2418 was found to cause a reduction in the adhesion of the bacteria to bronchial cells and mucins. Moreover, in vivo studies have shown that the anti-BCAM2418 antibody exerted an inhibitory effect during infection in Galleria mellonella. Finally, inferred by glycan arrays, we were able to predict for the first time, host glycan epitopes for a TAA. We show that BCAM2418 favoured binding to 3'sialyl-3-fucosyllactose, histo-blood group A, α-(1,2)-linked Fuc-containing structures, Lewis structures and GM1 gangliosides. In addition, the glycan microarrays demonstrated similar specificities of Burkholderia species for their most intensely binding carbohydrates.

Examination of oestrus-dependent alterations of bovine cervico-vaginal mucus glycosylation for potential as optimum fertilisation indicators.

Oestrus is the period in the sexual cycle of female mammals where they become most receptive to mating and are most fertile. Efficient detection of oestrus is a key component in successful reproductive livestock management programmes. Oestrus detection in cattle is most often performed by visual observation, such as mounting behaviour and standing heat, to facilitate more successful prediction of optimal time points for artificial insemination. This time-consuming method requires a skilled, diligent observer. Biological measurements using easily accessible biomolecules in the cervico-vaginal mucus could provide an alternative strategy to physical methods of oestrus detection, providing an inexpensive means of rapidly and accurately assessing the onset of oestrus. In this study, glycosylation changes in cervico-vaginal mucus from three heifers following oestrus induction were investigated as a proof of concept to assess whether potential glycosylation-based trends could be useful for oestrus stage indication. Mucus collected at different time points following oestrus induction was immobilised in a microarray format and its glycosylation interrogated with a panel of fluorescently labelled lectins, carbohydrate-binding proteins with different specificities. Individual animal-specific glycosylation patterns were observed, however each pattern followed a similar trend around oestrus. This unique oestrus-associated glycosylation was identified by a combination of relative binding of the lectins SNA-I and WFA for each animal. This alteration in cervico-vaginal mucus glycosylation could potentially be exploited in future to more accurately identify optimal fertilisation intervention points compared to visual signs. More effective oestrus biomarkers will lead to more successful livestock reproductive programmes, decreasing costs and animal stress.

Elastin-like recombinamers-based hydrogel modulates post-ischemic remodeling in a non-transmural myocardial infarction in sheep.

Ischemic heart disease is a leading cause of mortality due to irreversible damage to cardiac muscle. Inspired by the post-ischemic microenvironment, we devised an extracellular matrix (ECM)-mimicking hydrogel using catalyst-free click chemistry covalent bonding between two elastin-like recombinamers (ELRs). The resulting customized hydrogel included functional domains for cell adhesion and protease cleavage sites, sensitive to cleavage by matrix metalloproteases overexpressed after myocardial infarction (MI). The scaffold permitted stromal cell invasion and endothelial cell sprouting in vitro. The incidence of non-transmural infarcts has increased clinically over the past decade, and there is currently no treatment preventing further functional deterioration in the infarcted areas. Here, we have developed a clinically relevant ovine model of non-transmural infarcts induced by multiple suture ligations. Intramyocardial injections of the degradable ELRs-hydrogel led to complete functional recovery of ejection fraction 21 days after the intervention. We observed less fibrosis and more angiogenesis in the ELRs-hydrogel-treated ischemic core region compared to the untreated animals, as validated by the expression, proteomic, glycomic, and histological analyses. These findings were accompanied by enhanced preservation of GATA4+ cardiomyocytes in the border zone of the infarct. We propose that our customized ECM favors cardiomyocyte preservation in the border zone by modulating the ischemic core and a marked functional recovery. The functional benefits obtained by the timely injection of the ELRs-hydrogel in a clinically relevant MI model support the potential utility of this treatment for further clinical translation.