Structure Of Glycoprotein Research Articles

Cryo-EM structure of Sudan ebolavirus glycoprotein complexed with its human endosomal receptor NPC1

Sudan ebolavirus (SUDV), like Ebola ebolavirus (EBOV), poses a significant threat to global health and security due to its high lethality. However, unlike EBOV, there are no approved vaccines or treatments for SUDV, and its structural interaction with the endosomal receptor NPC1 remains unclear. This study compares the glycoproteins of SUDV and EBOV (in their proteolytically primed forms) and their binding to human NPC1 (hNPC1). The findings reveal that the SUDV glycoprotein binds significantly more strongly to hNPC1 than the EBOV glycoprotein. Using cryo-EM, we determined the structure of the SUDV glycoprotein/hNPC1 complex, identifying four key residues in the SUDV glycoprotein that differ from those in the EBOV glycoprotein and influence hNPC1 binding: Ile79, Ala141, and Pro148 enhance binding, while Gln142 reduces it. Collectively, these residue differences account for SUDV’s stronger binding affinity for hNPC1. This study provides critical insights into receptor recognition across all viruses in the ebolavirus genus, including their interactions with receptors in bats, their suspected reservoir hosts. These findings advance our understanding of ebolavirus cell entry, tissue tropism, and host range.

Methods for detecting, building, and improving tryptophan mannosylation in glycoprotein structures.

Tryptophan mannosylation, the covalent addition of an α-ᴅ-mannose sugar to a tryptophan side chain, is a post-translational modification (PTM) that can affect protein stability, folding, and interactions. Compared to other forms of protein glycosylation, it is relatively uncommon but is affected by conformational anomalies and modeling errors similar to those seen in N- and O-glycans in the Protein Data Bank (PDB). In this work, we report methods for detecting, building, and improving mannose structures linked to tryptophans. These methods have been used to mine X-ray crystallographic and cryo-electron microscopy maps in the PDB looking for unmodeled mannosylation, resulting in a number of cases where the modification can be placed in the map with high confidence. Additionally, we address most conformational issues affecting this modification. Finally, the development of a structural template to recognize thrombospondin repeats (TSR) domains where tryptophan mannosylation occurs will allow for the mannosylation of candidate-predicted models, for example, those predicted with AlphaFold.

SARS-CoV-2 S, M, and E Structural Glycoproteins Differentially Modulate Endoplasmic Reticulum Stress Responses

We have previously shown that the hepatitis C virus (HCV) E1E2 envelope glycoprotein can regulate HIV-1 long-terminal repeat (LTR) activity through disruption to NF-κB activation. This response is associated with upregulation of the endoplasmic reticulum (ER) stress response pathway. Here, we demonstrate that the SARS-CoV-2 S, M, and E but not the N structural protein can perform similar downmodulation of HIV-1 LTR activation, and in a dose-dependent manner, in both HEK293 and lung BEAS-2B cell lines. This effect is highest with the SARS-CoV-2 Wuhan S strain and decreases over time for the subsequent emerging variants of concern (VOC), with Omicron providing the weakest effect. We developed pseudo-typed viral particle (PVP) viral tools that allowed for the generation of cell lines constitutively expressing the four SARS-CoV-2 structural proteins and utilising the VSV-g envelope protein to deliver the integrated gene construct. Differential gene expression analysis (DGEA) was performed on cells expressing S, E, M, or N to determine cell activation status. Gene expression differences were found in a number of interferon-stimulated genes (ISGs), including IF16, IFIT1, IFIT2, and ISG15, as well as for a number of heat shock protein (HSP) genes, including HSPH1, HSPA6, and HSPBP1, with all four SARS-CoV-2 structural proteins. There were also differences observed in expression patterns of transcription factors, with both SP1 and MAVS upregulated in the presence of S, M, and E but not the N protein. Collectively, the results indicate that gene expression patterns associated with ER stress pathways can be activated by SARS-CoV-2 envelope glycoprotein expression. The results suggest the SARS-CoV-2 infection can modulate an array of cell pathways, resulting in disruption to NF-κB signalling, hence providing alterations to multiple physiological responses of SARS-CoV-2-infected cells.

The endocuticle structural glycoprotein AgSgAbd-2-like is required for cuticle formation and survival in the melon aphid Aphis gossypii.

Cuticular proteins are essential for cuticle formation, molting, and survival in insects. However, functional analysis of cuticular proteins in the melon aphid has been limited. In this study, we identified an endocuticle structural glycoprotein (ESG) AgSgAbd-2-like in the melon aphid Aphis gossypii, which is a member of the RR-1 subfamily of the CPR (cuticular protein containing the conserved Rebers-Riddiford motif) chitin-binding proteins. When double-stranded RNA is delivered epidermally, AgSgAbd-2-like is knocked down, resulting in molting defects and mortality. The expression of AgSgAbd-2-like is comparatively low prior to molting and increases following molting. Ecdysone signaling consistently suppresses AgSgAbd-2-like. Histologically, the endocuticle and whole cuticle are thinner in AgSgAbd-2-like RNA interference (RNAi) aphids, which is a leading cause of molting defects and mortality. Furthermore, knockdown of any other homolog of ESGs, including AgSgAbd-4, AgSgAbd-4-like, AgSgAbd-8-like, and AgSgAbd-9-like, results in molting defects and death, like that by AgSgAbd-2-like RNAi. These results indicate that the melon aphid ESGs are conserved in cuticle formation and could be potential targets for RNAi-based pest management.

A novel DUF 3472 domain-containing fern protein impairs reproduction in Helicoverpa armigera

Helicoverpa armigera is a polyphagous field crop insect pest. It poses a major threat to economically important crops leading to significant financial losses globally. Given the escalating resistance cases against chemical and Bt-based insecticides, there is an urgent need to identify new molecules to control this insect through different modes of action.In this endeavour, we have isolated a new protein [named Msc42] efficacious to H. armigera from the fern Microsorum scolopendria. The protein has two domains of unknown function- DUF 5077 and DUF 3472. At an LC50 of 3.6 μg/g, Msc42 severely impairs molting and metamorphosis in surviving larvae. Mass spectrometric analysis of the total soluble protein of larvae identified altered regulatory proteins responsible for impaired insect growth and reproduction. This includes larval cuticle proteins and endocuticle structural glycoproteins. Storage proteins were either at lower levels or below the detection threshold. Vitellogenins were also found deficient. The microscopic study showed that the fern protein ravaged ovarian follicle development leading to complete reproductive failure. Our results indicate that the novel fern protein Msc42 may offer an alternative strategy for controlling H. armigera.

Molecular imprinting resonant light scattering sensor based on teamed boronate affinity for highly specific detection of glycoprotein

It is challenging to detect and enrich glycoproteins in complex biological samples because they are low in natural abundance, highly flexible in conformation and there are many interfering substances in the samples. To overcome these challenges, magnetic Fe3O4 nanoparticles (NPs) were prepared, in which the surface was modified by metal–organic frameworks (MOFs) and then by molecularly imprinted polymers (MIPs). Combined with the teamed boronate affinity (TBA) strategy, the selectivity and sensitivity detection of glycoprotein ovalbumin (OVA) were realized. The magnetic Fe3O4 NPs were coated with MOFs to facilitate the adsorption of AuNPs, onto which the TBA moieties composed of 2-mercaptoethylamine and 4-mercaptophenylboronic acid were pre-assembled. The final magnetic Fe3O4@TBA/MIPs NPs featured shallow imprinted cavities, resulting in rapid mass transfer of the target glycoprotein (equilibrium time 20 min). In particular, the TBA strategy relied on N-B synergy, resulting in high specificity (IF = 4.52) and high sensitivity (limit of detection 13 pM) for the detection of OVA at physiological pH (7.4). This strategy provides an extremely convenient method for detecting and quantifying low-abundance glycoprotein in complex biological samples, without destroying the structure and function of the target glycoprotein.

Mechanistic and Therapeutic Implications of Protein and Lipid Sialylation in Human Diseases.

Glycan structures of glycoproteins and glycolipids on the surface glycocalyx and luminal sugar layers of intracellular membrane compartments in human cells constitute a key interface between intracellular biological processes and external environments. Sialic acids, a class of alpha-keto acid sugars with a nine-carbon backbone, are frequently found as the terminal residues of these glycoconjugates, forming the critical components of these sugar layers. Changes in the status and content of cellular sialic acids are closely linked to many human diseases such as cancer, cardiovascular, neurological, inflammatory, infectious, and lysosomal storage diseases. The molecular machineries responsible for the biosynthesis of the sialylated glycans, along with their biological interacting partners, are important therapeutic strategies and targets for drug development. The purpose of this article is to comprehensively review the recent literature and provide new scientific insights into the mechanisms and therapeutic implications of sialylation in glycoproteins and glycolipids across various human diseases. Recent advances in the clinical developments of sialic acid-related therapies are also summarized and discussed.

Structures of the Varicella Zoster Virus Glycoprotein E and Epitope Mapping of Vaccine-Elicited Antibodies.

Background: Varicella zoster virus (VZV) is the causative agent for chickenpox and herpes zoster (HZ, shingles). HZ is a debilitating disease affecting elderly and immunocompromised populations. Glycoprotein E (gE) is indispensable for viral replication and cell-to-cell spread and is the primary target for anti-VZV antibodies. Importantly, gE is the sole antigen in Shingrix, a highly efficacious, AS01B-adjuvanted vaccine approved in multiple countries for the prevention of HZ, yet the three-dimensional (3D) structure of gE remains elusive. Objectives: We sought to determine the structure of VZV gE and to understand in detail its interactions with neutralizing antibodies. Methods: We used X-ray crystallography and cryo-electron microscopy to elucidate structures of gE bound by recombinant Fabs of antibodies previously elicited through vaccination with Zostavax, a live, attenuated vaccine. Results: The 3D structures resolve distinct central and C-terminal antigenic domains, presenting an array of diverse conformational epitopes. The central domain has two beta-sheets and two alpha helices, including an IgG-like fold. The C-terminal domain exhibits 3 beta-sheets and an Ig-like fold and high structural similarity to HSV1 gE. Conclusions: gE from VZV-infected cells elicits a human antibody response with a preference for the gI binding domain of gE. These results yield insights to VZV gE structure and immunogenicity, provide a framework for future studies, and may guide the design of additional herpesvirus vaccine antigens. Teaser: Structures of varicella zoster virus glycoprotein E reveal distinct antigenic domains and define epitopes for vaccine-elicited human antibodies.

The roles of rabies virus structural proteins in immune evasion and implications for vaccine development.

Rabies is a zoonotic infectious disease that targets the nervous system of human and animals and has about 100% fatality rate without treatment. Rabies virus is a bullet-like viral particle composed of five structural proteins, including nucleoprotein (N), phosphorylated protein (P), matrix protein (M), glycoprotein (G), and large subunit (L) of RNA-dependent RNA polymerase. These multifunctional viral proteins also play critical roles in the immune escape by inhibiting specific immune responses in the host, resulting in massive replication of the virus in the nervous system and abnormal behaviors of patients such as brain dysfunction and hydrophobia, which ultimately lead to the death of patients. Herein, the role of five structural proteins of rabies virus in the viral replication and immune escape and its implication for the development of vaccines were systemically reviewed, so as to shed light on the understanding of pathogenic mechanism of rabies virus.

Research progress on the structure and function of the main glycoproteins of bovine infectious rhinotracheitis virus

Research progress on the structure and function of the main glycoproteins of bovine infectious rhinotracheitis virus

Mapping glycoprotein structure reveals Flaviviridae evolutionary history

Viral glycoproteins drive membrane fusion in enveloped viruses and determine host range, tissue tropism and pathogenesis1. Despite their importance, there is a fragmentary understanding of glycoproteins within the Flaviviridae2, a large virus family that include pathogens such as hepatitis C, dengue and Zika viruses, and numerous other human, animal and emergent viruses. For many flaviviruses the glycoproteins have not yet been identified, for others, such as the hepaciviruses, the molecular mechanisms of membrane fusion remain uncharacterized3. Here we combine phylogenetic analyses with protein structure prediction to survey glycoproteins across the entire Flaviviridae. We find class II fusion systems, homologous to the Orthoflavivirus E glycoprotein in most species, including highly divergent jingmenviruses and large genome flaviviruses. However, the E1E2 glycoproteins of the hepaciviruses, pegiviruses and pestiviruses are structurally distinct, may represent a novel class of fusion mechanism, and are strictly associated with infection of vertebrate hosts. By mapping glycoprotein distribution onto the underlying phylogeny, we reveal a complex evolutionary history marked by the capture of bacterial genes and potentially inter-genus recombination. These insights, made possible through protein structure prediction, refine our understanding of viral fusion mechanisms and reveal the events that have shaped the diverse virology and ecology of the Flaviviridae.

Arabinosylation of cell wall extensin is required for the directional response to salinity in roots.

Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploited a real-time salt-induced tilting assay. This assay follows root growth upon both gravitropic and salt challenges, revealing that root bending upon tilting is modulated by Na+ ions, but not by osmotic stress. Next, we measured this salt-specific response in 345 natural Arabidopsis (Arabidopsis thaliana) accessions and discovered a genetic locus, encoding the cell wall-modifying enzyme EXTENSIN ARABINOSE DEFICIENT TRANSFERASE (ExAD) that is associated with root bending in the presence of NaCl (hereafter salt). Extensins are a class of structural cell wall glycoproteins known as hydroxyproline (Hyp)-rich glycoproteins, which are posttranslationally modified by O-glycosylation, mostly involving Hyp-arabinosylation. We show that salt-induced ExAD-dependent Hyp-arabinosylation influences root bending responses and cell wall thickness. Roots of exad1 mutant seedlings, which lack Hyp-arabinosylation of extensin, displayed increased thickness of root epidermal cell walls and greater cell wall porosity. They also showed altered gravitropic root bending in salt conditions and a reduced salt-avoidance response. Our results suggest that extensin modification via Hyp-arabinosylation is a unique salt-specific cellular process required for the directional response of roots exposed to salinity.

Potential contribution of alpha-fetoprotein level to biomarker of pregnancy outcome in Asian elephants.

Alpha-fetoprotein (AFP) is a structural serum glycoprotein that plays vital roles in reproduction and mammalian development. Analysis of serum prolactin (PRL) is considered one of the useful methods for diagnosing pregnancy in Asian elephants. However, the expression profiles of AFP in pregnant and nonpregnant Asian elephants remain unclear, nor is the relationship with PRL. In this study, serum seven gonadal hormones and AFP in three pregnant and seven nonpregnant Asian elephants were analysed by via radioimmunoassay (RIA) and enzyme-linked immunosorbent (ELISA) assay. We found that the mean (±SD) concentration of prolactin (PRL) in pregnant (136.782 ± 30.987ng/mL) elephants was significantly higher than that in nonpregnant elephants (52.803 ± 21.070ng/mL; p ≤ 0.0005). The mean (±SD) concentration of AFP in pregnant elephants (11.598 ± 0.824ng/mL) was significantly higher than that in nonpregnant elephants (7.200 ± 2.283ng/mL; p ≤ 0.05). Furthermore, the AFP concentration was positively correlated with the PRL concentration in the 10 Asian elephants studied. In conclusion, our findings suggest that serum AFP concentration is a potential biomarker of pregnancy outcomes in Asian elephants.

Sweet Secrets: Exploring Novel Glycans and Glycoconjugates in the Extracellular Polymeric Substances of "Candidatus Accumulibacter".

Biological wastewater treatment relies on microorganisms that grow as flocs, biofilms, or granules for efficient separation of biomass from cleaned water. This biofilm structure emerges from the interactions between microbes that produce, and are embedded in, extracellular polymeric substances (EPS). The true composition and structure of the EPS responsible for dense biofilm formation are still obscure. We conducted a bottom-up approach utilizing advanced glycomic techniques to explore the glycan diversity in the EPS from a highly enriched "Candidatus Accumulibacter" granular sludge. Rare novel sugar monomers such as N-Acetylquinovosamine (QuiNAc) and 2-O-Methylrhamnose (2-OMe-Rha) were identified to be present in the EPS of both enrichments. Further, a high diversity in the glycoprotein structures of said EPS was identified by means of lectin based microarrays. We explored the genetic potential of "Ca. Accumulibacter" high quality metagenome assembled genomes (MAGs) to showcase the shortcoming of top-down bioinformatics based approaches at predicting EPS composition and structure, especially when dealing with glycans and glycoconjugates. This work suggests that more bottom-up research is necessary to understand the composition and complex structure of EPS in biofilms since genome based inference cannot directly predict glycan structures and glycoconjugate diversity.

Rational design and computational evaluation of a multi-epitope vaccine for monkeypox virus: Insights into binding stability and immunological memory

Multi-epitope vaccines strategically tackle rapidly mutating viruses by targeting diverse epitopes from different proteins, providing a comprehensive and adaptable immune protection approach for enhanced coverage against various viral variants. This research employs a comprehensive approach that includes the mapping of immune cells activating epitopes derived from the six structural glycoproteins (A29L, A30L, A35R, L1R, M1R, and E8L) of Monkeypox virus (Mpox). A total of 7 T-cells-specific epitopes, 13 B-cells-specific epitopes, and 5 IFN-γ activating epitopes were forecasted within these glycoproteins. The selection process focused on epitopes indicating high immunogenicity and favorable binding affinity with multiple MHC alleles. Following this, a vaccine has been formulated by incorporating the chosen epitopes, alongside adjuvants (PADRE peptide) and various linkers (EAAAK, GPGPG, and AAY). The physicochemical properties and 3D structure of the multi-epitope hybrid vaccine were analysed for characterization. MD simulations were employed to predict the binding stability between the vaccine and various pathogen recognition receptors such as TLRs (TLR1, TLR2, TLR4, and TLR6), as well as both class I and II MHC, achieved through hydrogen bonding and hydrophobic interactions. Through in silico cloning and immune simulation, it was observed that the multi-epitopes vaccine induced a robust memory immune response upon booster doses, forecasting protective immunity upon viral challenge. This protective immunity was characterized by the production of IgM + IgG antibodies, along with release of inflammatory cytokines like IFN-γ, and IL12, and the activation of various immune cells. This study offers valuable insights into the potential of a multi-epitope vaccine targeting the Mpox virus.

A natural compound from plant, Thailandiol as a potential dual inhibitor for spike glycoprotein, and RBD region of ACE2-spike glycoprotein: an insilico ADMET analysis and molecular docking study

COVID-19 primarily targets the angiotensin-converting enzyme 2 (ACE2) receptor, which enables the virus to infiltrate host cells via the spike glycoprotein. In this research, Thailandiol could serve as a dual inhibitor for the spike glycoprotein and the interface of the spike glycoprotein and ACE2. This was achieved using molecular docking and ADMET analysis. We docked Thailandiol against the crystal structures of the spike glycoprotein and the Receptor Binding Domain region of SARS-CoV-2 and the ACE2 receptor using AutoDock4 & Vina. Thailandiol demonstrated high binding affinity and favourable interactions with both the spike glycoprotein and the RBD region. Additionally, Thailandiol exhibited favourable ADMET properties and Swiss target prediction. Our in-silico research indicates that Thailandiol has the potential to be a dual inhibitor for the spike glycoprotein and the interface of the RBD region, and could be developed into a novel therapeutic agent for COVID-19.

Synthesis of Cobalt(III) Complexes Derived from Pyridoxal: Structural Cleavage Evaluations and In Silico Calculations for Biological Targets

This study sought to investigate the synthesis of eight complexes constituted by a cobalt(III) (CoIII) metallic center coordinated to two units of iminic ligands LnC (n = 1–4, L1C–L4C), which are derivatives of pyridoxal hydrochloride and anilines with thioether function containing one to four carbons. Depending on the source of the cobalt ion and the addition (or not) of a non-coordinating counterion, complexes with distinct structures may form, being categorized into two series: [CoIII(LnC)(L0C)] (n = 1–4, C1’–C4’) with a LnC ligand and a ligand that has a thiolate function which cleaves the C-S(thioether) bond (L0C) and [CoIII(LnC)2]PF6 (n = 1–4, C1–C4) with two similar units of the same LnC ligand. The occurrence (or not) of cleavage in the eight complexes was observed by elucidating the solid-state structures by single crystal X-ray diffraction. This exciting method allows the synthesis of CoIII complexes without cleaving the C-S bonds from the ligands, thereby not requiring an inert atmosphere in the reaction systems. The synthesized complexes were evaluated by in silico calculations on viable biological targets such as deoxyribonucleic acid, superoxide dismutase enzyme, human serum albumin, and the structural spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the receptor binding domain (RBD) in both up and down conformations without and in complex with the cellular receptor angiotensin-converting enzyme 2 (ACE2). Overall, in silico results suggested that all the inorganic complexes under study are potential anticancer/antiviral agents; however, C4 and C4’ are the best candidates for future in vitro assays.

Extracellular vesicles in cattle infected with bovine leukaemia virus: isolation and molecular analysis.

Exosomes are nanosized lipid bilayer membranous microvesicles, extracellularly released from a variety of mammalian cells. They mediate intercellular signalling by transporting several types of RNA, lipids and proteins and participate in the intercellular exchange of DNA, RNA, micro RNA, proteins and other components. These microvesicles are present in all body fluids in physiological and pathological conditions and reflect the state of the host organism. The aim of the study was the isolation and molecular determination of exosomes in blood and supernatant fluids of bovine dendritic cell cultures infected with bovine leukaemia virus (BLV). Exosomes were isolated by ultracentrifugation from the blood sera, plasma and supernatant of bovine BLV-infected and uninfected control dendritic cell cultures and their presence was confirmed with scanning electron and transmission electron microscopy. Western blot analysis of the structural BLV glycoprotein 51 (Env) and protein 24 (Gag) and of the tetraspanin exosomal markers CD9, CD63 and flotillin-1 was undertaken in BLV+ and control BLV- cattle. In exosomes of leukaemic cattle both BLV proteins and exosomal markers were detected. In healthy control animals only exosomal markers were determined. Proteins of BLV were released with exosomes and could be transferred into recipient cells as an alternative propagation route not requiring virus infection.

Erythroid anion transport, nitric oxide, and blood pressure.

Glycophorin A and glycophorin B are structural membrane glycoproteins bound in the band 3 multiprotein complexes on human red blood cells (RBCs). Band 3 is an erythroid-specific anion exchanger (AE1). AE1-mediated HCO3 - transport provides the substrate for the enzyme-catalyzed conversion HCO3 - (aq) ⇌ CO2(g), which takes place inside the RBCs. Bicarbonate transport via AE1 supports intravascular acid-base homeostasis and respiratory excretion of CO2. In the past decade, we conducted several comparative physiology studies on Taiwanese people having the glycophorin variant GPMur RBC type (which accompanies greater AE1 expression). We found that increased anion transport across the erythrocyte membrane not only enhances gas exchange and lung functions but also elevates blood pressure (BP) and reduces nitric oxide (NO)-dependent vasodilation and exhaled NO fraction (FeNO) in healthy individuals with GP.Mur. Notably, in people carrying the GPMur blood type, the BP and NO-dependent, flow-mediated vasodilation (FMD) are both more strongly correlated with individual hemoglobin (Hb) levels. As blood NO and nitrite (NO2 -) are predominantly scavenged by intraerythrocytic Hb, and NO2 - primarily enters RBCs via AE1, could a more monoanion-permeable RBC membrane (i.e., GPMur/increased AE1) enhance NO2 -/NO3 - permeability and Hb scavenging of NO2 - and NO to affect blood pressure? In this perspective, a working model is proposed for the potential role of AE1 in intravascular NO availability, blood pressure, and clinical relevance.

Citric acid tailors the mechanical and barrier properties of arabinoxylan-gluten crosslinked glycoprotein films

Side streams from wheat processing constitute a widely available renewable resource from biomass rich in carbohydrate polymers and proteins, with great potential as biopolymeric matrices for the production of biobased films for food packaging applications. In this study, arabinoxylan (AX) was extracted with high purity from wheat bran via alkaline extraction and was combined with wheat gluten for the preparation of AX-gluten glycoprotein films using citric acid (CA) as a crosslinker and glycerol as plasticizer. Self-standing and flexible films were prepared via solvent casting and further cured at 150 °C to initiate crosslinking. FTIR analysis and solubility tests revealed that chemical crosslinking had occurred to some extent involving both the AX and gluten components, suggesting the formation of glycoprotein structures. However, the evaluation of the mechanical and barrier properties of the films showed that CA mainly works as a plasticizing agent rather than a crosslinking agent at the conditions used in the study. UV–Vis spectroscopy measurements showed that the films had an excellent ability to block UV-light. This study demonstrates the successful preparation of AX-gluten glycoprotein films with excellent mechanical and barrier properties, which can be tuned by the protein content and CA addition balancing both plasticizing and crosslinking effects.