Wheat Germ Agglutinin Research Articles

Oxytocin attenuates cardiac hypertrophy by improving cardiac glucose metabolism and regulating OXTR/JAK2/STAT3 axis

BackgroundThe progress of cardiac hypertrophy is modulated by JAK2/STAT3 signaling pathway. Cardiac glucose metabolism derangement exacerbates the progression of cardiac hypertrophy. Oxytocin (OT) has emerged as a significant hormone involved in cardiovascular homeostasis, especially in protecting against cardiac hypertrophy. The present study aims to explore whether the anti-hypertrophy effect of oxytocin is related to the JAK2/STAT3 signaling pathway and cardiac glucose metablism. MethodsCardiac hypertrophy model was induced by angiotensin II (Ang II) in H9c2 cells and in mice with or without oxytocin treatment. Changes in cardiac histopathology were evaluated by hematoxylin and eosin (H&E), Masson staining, and wheat germ agglutinin (WGA) staining. The hypertrophy-related genes and JAK2/STAT3 pathway signaling molecules were analyzed by qRT-PCR and western blotting. The levels of glucose, pyruvic acid, lactic acid, and lactate dehydrogenase activity in H9c2 cells using the corresponding assay kits. ResultsThe results showed that OT inhibited hypertrophic and fibrotic changes. Furthermore, OT increased intracellular levels of glucose and pyruvic acid, and decreased lactate dehydrogenase activity and lactic acid levels. Mechanistically, Ang II decreased oxytocin receptors (OXTR) expression and facilitated JAK2 and STAT3 phosphorylation. OT treatment increased OXTR expression and blocked JAK2 and STAT3 phosphorylation The OXTR-specific siRNA-mediated depleted expression could abrogate OT-induced anti-hypertrophic effects in H9c2 cells following angiotensin II insult. However, the JAK2/STAT3 inhibitor AG490 rescued the protective effects of OT against cardiac hypertrophy under OXTR downregulation. Conclusion:OT exerts its protective effects against cardiac hypertrophy by improving cardiac glucose metabolism and regulating OXTR/JAK2/STAT3 axis.

Glycosylation changes of vWF in circulating extracellular vesicles to predict depression

The clinical diagnosis of major depressive disorder (MDD) still depends on subjective information in terms of various symptoms regarding mood. Detecting the characterization of extracellular vesicles (EVs) in blood may result in finding a diagnostic biomarker that reflects the depressive stage of patients with MDD. Here, we report the results on the glycosylation pattern of enriched plasma EVs from patients with MDD. We compared glycosylation patterns by lectin blotting expressed in EVs isolated from the plasma of both patients with MDD and age-matched healthy control participants (HCs) using size-exclusion chromatography. The levels of Wheat germ agglutinin (WGA), N-acetyl glucosamine (GlcNAc), and N-Acetylneuraminic acid (Neu5Ac, sialic acid) - binding lectin, were significantly decreased in patients with MDD in the depressive state compared to HCs and in remission state. Furthermore, proteome analysis revealed that the von Willebrand factor (vWF) was a significant factor recognized by WGA. WGA-binding vWF antigen differentiated patients with MDD versus HCs and the same patients with MDD in a depressive versus remission state. In this study, the change patterns in the glycoproteins contained in plasma EVs support the usability of testing to identify patients who are at increased risk of depression during antidepressant treatment.

Perfusion staining methods for visualization of the intact microvascular networks in whole mount skeletal muscle preparations.

Visualization of the intact microvascular network in skeletal muscle requires labeling the entire network in whole mount preparations where muscle fibre length can be set to near optimal but the tools to do this are not clear. We intravascularly injected CD-1 mice with different fluorescently labelled lectins (fluorescent isolectin GS-IB4 (ISO), wheat germ agglutinin (WGA), lycopersicon esculentum (tomato) (LYCO) or FITC-labelled gel. Soleus, extensor digitorum longus, diaphragm, gluteus maximus and cremaster muscles were excised, pinned at optimal sarcomere length and viewed using fluorescence microscopy. WGA and LYCO were the effective at labeling the entire vascular network with WGA labeling capillaries more bright. ISO labelled the arteriolar vasculature and early segments of the capillaries but not the full length of the capillaries or the venular network. FITC-labelled gel was effective at labelling the microvascular network but not all small vessels were consistently labelled. The pattern of staining for each labelling method was similar across all muscle fibre-types tested. WGA was optimal for perfusion labeling and visualization of the intact microvascular network in whole mount skeletal muscle preparations and can be used in combination with ISO to distinguish the arteriolar and venous sides of the network.

CardiLect: A combined cross-species lectin histochemistry protocol for the automated analysis of cardiac remodelling.

Cardiac remodelling, a crucial aspect of heart failure, is commonly investigated in preclinical models by quantifying cardiomyocyte cross-sectional area (CSA) and microvascular density (MVD) via histological methods, such as immunohistochemistry. To achieve this, optimized protocols are needed, and the species specificity is dependent on the antibody used. Lectin histochemistry offers several advantages compared to antibody-based immunohistochemistry, including as cost-effectiveness and cross-species applicability. Direct comparisons between the two methods are lacking from the literature. In this study, we compared antibody- and lectin-based methods for the histological assessment of cardiomyocyte CSA (with the use of anti-laminin and wheat germ agglutinin [WGA]) and microvascular density (utilizing anti-CD31 and isolectin B4 [ILB4]) using different embedding and antigen/carbohydrate retrieval techniques. Here, we describe a detailed, easy-to-use combined lectin histochemistry protocol (WGA and ILB4, 'CardiLect' protocol) for the histological assessment of cardiac remodelling. The lectin-based approach has been evaluated on a cross-species basis, and its efficacy has been demonstrated in zebrafish, rodents, large animals and human samples. We provide an ImageJ script ('CardiLect Analyser') for automated image analysis, validated in a preclinical heart failure model by correlating histological parameters with echocardiographic findings. CSA showed a significant positive correlation with left ventricular (LV) mass (P=0.0098, rS=0.7545) and significant negative correlation with markers of systolic function, such as ejection fraction (EF) (P=0.0402, rS=-0.6364). Microvascular density showed significant negative correlation with LV mass (P=0.0055, rS=-0.7622) and significant positive correlation with EF (P=0.0106, rS=0.7203). The described combined lectin histochemistry protocol with the provided ImageJ script is an easy-to-use, cost-effective, cross-species approach for the histological assessment of cardiac remodelling.

Abstract 4136700: Sodium-Potassium Pump: Newly O-GlcNAc Protein Offering Potential Benefits in Hemorrhagic Shock situation

Hemorrhagic shock(HS) results from significant blood loss, leading to insufficient oxygen delivery, which causes metabolic changes and multi-organ dysfunction.The HS-related acute kidney injury developed by the patient are mainly due to alteration in Na/K ATPase.In previous O-GlcNAcylomic studies, we identified the Na/K ATPase as potentially O-GlcNAcylated.O-GlcNAcylation, a post-translational modification, plays a role in various cellular processes, such as cell survival and stress response.O-GlcNAcylation may have beneficial effect in shock situations. The objective of this study is to ascertain whether Na/K ATPase is O-GlcNAcylated and to elucidate its potential role during HS. Hemorrhagic shock was induced by an exsanguination of rats.They were then randomly treated or not with an O-GlcNAcase inhibitor, the NButGT(10mg/kg), to increase O-GlcNAc level.Blood pressure and heart rate were monitored throughout the experiment.Blood samples were collected for complete blood analysis, including biochemistry and gas measurements. kidneys were immediately frozen for histological and biochemical studies.To study the O-GlcNAcylation of Na/K ATPase an immunoprecipitation and Wheat Germ Agglutinin(WGA) based lectin affinity gel electrophoresis were used. HEK cells were treated with or without NButGT(100µM,6 hours) and/or ouabain(Na/K ATPase inhibitor) and a colorimetric kit was assessed to study O-GlcNAcylation impact on Na/K ATPase activity. O-GlcNAc levels increased by 2 folds in heart and kidney due to the NButGT treatment(p<0.05).Mean arterial pressure(MAP,p<0.01) and bicarbonate(p<0.01) are restored in treated group as kalemia(Sham:4.2 ± 0.1;HS:4.7±0.1;NButGT:4.3 ±0.1;mmol/L;p<0.05) and natremia(Sham:141.5±0.4;HS:139.8±0.5;NButGT:141.9 ±0.4; mmol/L;p<0.01).The study of the O-GlcNAcylation of the Na/K ATPase validated its O-GlcNAcylation.Histological analysis of the kidney showed that HS decreased Na/K ATPase expression, but treatment restored it(Sham:15.1±1.1;HS:10.3±0.6;NButGT:13.78±1.1;%area;p<0.05). An acute increase in O-GlcNAc levels through NButGT enhances Na/K ATPase activity. Increased O-GlcNAcylation during hemorrhagic shock restored MAP and reduced indicators of metabolic acidosis.Treatment with NButGT restored natremia and kalemia.This effect is linked to an increase in the expression and activity of Na/K ATPase.Our study is the first to confirm that Na/K ATPase is regulated by O-GlcNAcylation, particularly in terms of its localization and stability.

Abstract 4114625: Upregulation of Palmitoylating Enzymes, DHHC2 and DHHC5, in Failing Human Hearts Protects Impulse Propagation but Endangers Myocyte Survival after Ischemia/Reperfusion (I/R) Injury

Background: Palmitoylation 'Palm* catalyzed by DHHC enzymes promotes protein binding to membranes, impacting protein translocation and function. Palm* dysregulation has been linked to cancers and neurogenerative disorders; its impact on cardiac function is unclear. We have used global purification of palmitoylated proteins from human and animal hearts followed by proteomic analysis to build 'cardiac palmitoylome', whose 454 constituents are involved in organizing/stabilizing cardiomyocyte microdomains (intercalated discs 'ICD', dyads, and costameres) critical for myocyte functions/homeostasis. We confirmed 11 of the 23 DHHC genes are expressed in human&animal hearts. In particular, DHHC2 and DHHC5 were upregulated in human failing hearts. DHHC enzymes are membrane-embedded, limiting their substrates to enzymes' 'territories' in cells. Objective: To identify DHHC2 and DHHC5 distribution patterns and substrates in myocytes, and to understand how upregulation of DHHC2 or DHHC5 affects myocyte function. Methods: Track adenovirus-expressed mCherry-DHHC2 and -DHHC5 (mChr-DHHCx, x=2 or 5, Adv-mChr as control) in ventricular myocytes from adult Sprague-Dawley rats. Globally purify palmitoylated proteins for proteomic analysis/quantification. Use high-resolution quantitative imaging to analyze how mChr-DHHCx affects myocyte response to ischemia/reperfusion (I/R) injury and the expression/organization of palmitoylated proteins at ICD and lateral surface. Results: mChr-DHHC2 is mainly present in cytoplasmic vesicles bearing markers of recycling or autophagy, with a minor presence along lateral surface, dyads, and ICD. mChr-DHHC5 is mainly present along the lateral surface, dyads and ICD, with a minor presence in small endosomal vesicles. Palmitoylome normalized by proteome identifies DHHC2 and DHHC5 substrates that overlap with cardiac palmitoylome, and match the enzymes' distribution patterns. I/R injury increases endocytosis in myocytes, quantified by uptake of plasma-membrane marker, wheat germ agglutinin. I/R-induced endocytosis is exacerbated by mChr-DHHC2 but, surprising, alleviated by mChr-DHHC5. mChr-DHHC2 and -DHHC5 increase internalization of ICD and lateral membrane proteins, n-cadherin and NCX1 respectively. Conclusions: DHHC2 and DHHC5 have non-overlapping distribution patterns, consistent with varying substrate groups. DHHC2, but not DHHC5, appears involved in the previously reported palm*-dependent massive endocytosis in I/R-stressed cardiomyocytes.

Inner Back Cover Image

ON THE INNER BACK COVER: Cross section of retinal photoreceptors. Green: outer segments (wheat germ agglutinin); red: actin filaments in the inner segment and outer segment base (phalloidin); blue: nuclei.Credit for Mohona Gupta and Gregory Pazour (Program of Molecular Medicine, University of Massachusetts, Chan Medical School). image

Early expression of osteopontin glycoprotein on the ocular surface and in tear fluid contributes to ocular surface diseases in type 2 diabetic mice.

Osteopontin (OPN) is a glycosylated, secreted phosphoprotein known to be elevated in both human and mouse retinas during various stages of diabetic retinopathy. However, its specific roles in modulating ocular surface dynamics and immune responses in diabetes remain unexplored. This study aims to investigate the role of OPN in the development of ocular surface disease (OSD) in type 2 diabetic (T2D) mice. Three- to four-week-old C57BL/6 wild-type (WT) and OPN-knockout (OPN-/-) mice were fed a high-fat diet (HFD) and were rendered diabetic by streptozotocin (STZ; 40 mg/kg body weight) in citrate buffer (vehicle); non-diabetic controls were injected with vehicle alone. Diabetes was confirmed if blood glucose levels were >200 mg/dL, measured 1-2 weeks post-STZ injection. Control, age- and sex-matched db/db diabetic mice fed a standard chow diet were also included in this study. Ocular surface inflammation was assessed using ELISA to quantify inflammatory cytokine proteins and wheat germ agglutinin (WGA) staining was utilized to highlight corneal surface irregularities. Clinical signs were evaluated by corneal fluorescein staining, tear production measurements, and tear sodium (Na+) concentration assessments. These evaluations were conducted 4, 6, 8 and 16-weeks post-diabetes onset in WT and OPN-/- mice and were compared to those obtained in non-diabetic controls. Statistical analysis was performed using a two-way ANOVA, with significance set at P < 0.05. Both WT and OPN-/- mice developed T2D within 4 and 8 weeks, respectively, following HFD + STZ treatment. Corneal OPN levels in WT diabetic mice increased ~2-fold at 2 weeks and ~4-fold at 16 weeks compared to non-diabetic controls, with similar elevations observed in their tear fluid. Diabetic db/db mice also exhibited elevated OPN levels in the blood and ocular surface, which persisted as diabetes progressed. Enhanced fluorescein staining, indicating corneal irregularities, appeared in WT mice at 8 weeks and in OPN-/- mice at 10 weeks post-T2D induction. Additionally, WGA staining showed a significant reduction in fluorescence intensity in WT mice treated with HFD and STZ, confirming corneal surface irregularities that were delayed in OPN-/- mice. Elevated tear sodium concentration was observed in both WT and OPN-/- diabetic mice without affecting tear production rates. Notably, OPN levels increased early, at week 2, following HFD and STZ treatment, preceding changes in interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and matrix metalloproteinase-9 (MMP-9). Upregulation of IL-6 became apparent at 6 weeks in WT mice and was delayed until 10 weeks in OPN-/- mice post-T2D induction. Elevated OPN levels were detected early post-T2D induction in diabetic WT and db/db mice corneas without initial subclinical changes. This early increase in OPN precedes other proinflammatory cytokines associated with eventual ocular surface inflammation as diabetes progresses. Persistence of OPN also correlated with clinical signs such as increased corneal surface irregularities and elevated tear Na+ concentration. Future research will explore OPN's role as a biomarker in ocular surface disease (OSD), including dry eye disease (DED), and investigate its impact on inflammatory processes and other mechanistic pathways in diabetic ocular complications.

Ultraviolet laser induced periodic surface structures positively influence osteogenic activity on titanium alloys.

Endoprostheses might fail due to complications such as implant loosening or periprosthetic infections. The surface topography of implant materials is known to influence osseointegration and attachment of pathogenic bacteria. Laser-Induced Periodic Surface Structures (LIPSS) can improve the surface topography of orthopedic implant materials. In this preclinical in vitro study, laser pulses with a wavelength in the ultraviolet (UV) spectrum were applied for the generation of LIPSS to positively influence formation of extracellular matrix by primary human Osteoblasts (hOBs) and to reduce microbial biofilm formation in vitro. Laser machining was employed for generating UV-LIPSS on sample disks made of Ti6Al4V and Ti6Al7Nb alloys. Sample disks with polished surfaces were used as controls. Scanning electron microscopy was used for visualization of surface topography and adherent cells. Metal ion release and cellular metal levels were investigated by inductively coupled plasma mass spectrometry. Cell culture of hOBs on sample disks with and without UV-LIPSS surface treatments was performed. Cells were investigated for their viability, proliferation, osteogenic function and cytokine release. Biofilm formation was facilitated by seeding Staphylococcus aureus on sample disks and quantified by wheat germ agglutinin (WGA) staining. UV-LIPSS modification results in topographies with a periodicity of 223nm ≤ λ ≤ 278nm. The release of metal ions was found increased for UV-LIPSS on Ti6Al4V and decreased for UV-LIPSS on Ti6Al7Nb, while cellular metal levels remain unaffected. Cellular adherence was decreased for hOBs on UV-LIPSS Ti6Al4V when compared to controls while proliferation rate was unaffected. Metabolic activity was lower on UV-LIPSS Ti6Al7Nb when compared to the control. Alkaline phosphatase activity was upregulated for hOBs grown on UV-LIPSS on both alloys. Less pro-inflammatory cytokines were released for cells grown on UV-LIPSS Ti6Al7Nb when compared to polished surfaces. WGA signals were significantly lower on UV-LIPSS Ti6Al7Nb indicating reduced formation of a S. aureus biofilm. Our results suggest that UV-LIPSS texturing of Ti6Al7Nb positively influence bone forming function and cytokine secretion profile of hOBs in vitro. In addition, our results indicate diminished biofilm formation on UV-LIPSS treated Ti6Al7Nb surfaces. These effects might prove beneficial in the context of long-term arthroplasty outcomes.

Establishment of a dexamethasone-induced zebrafish skeletal muscle atrophy model and exploration of its mechanisms

BackgroundSkeletal muscle atrophy is one of the main side effects of high-dose or continuous use of glucocorticoids (such as dexamethasone). However, there are limited studies on dexamethasone-induced skeletal muscle atrophy in zebrafish and even fewer explorations of the underlying molecular mechanisms. This study aimed to construct a model of dexamethasone-induced skeletal muscle atrophy in zebrafish and to investigate the molecular mechanisms. MethodsZebrafish soaked in 0.01 % dexamethasone solution for 10 days. Loli Track (Denmark) and Loligo Swimming Respirometer were used to observe the effect of dexamethasone on swimming ability. The effects of dexamethasone on zebrafish skeletal muscle were observed by Transmission electron microscopy, H&E, and wheat germ agglutinin techniques. Enriched genes and signaling pathways were analyzed using Transcriptome sequencing. Further, the levels of mitochondrial and endoplasmic reticulum-related proteins were examined to investigate possible mechanisms. Results0.01 % dexamethasone reduced zebrafish skeletal muscle mass (p < 0.05), myofibre size and cross-sectional area (p < 0.001), and increased protein degradation (ubiquitination and autophagy) (p < 0.05). In addition, 0.01 % dexamethasone reduced the swimming ability of zebrafish, as evidenced by the reluctance to move, fewer movement trajectories, decreased total distance traveled (p < 0.001), average velocity of movement (p < 0.001), oxygen consumption (p < 0.001), critical swimming speed (p < 0.01) and increased exhaustive swimming time (p < 0.001). Further, 0.01 % dexamethasone-induced mitochondrial dysfunction (decreased mitochondrial biogenesis, disturbs kinetic homeostasis, increased autophagy) and endoplasmic reticulum stress. Conclusions0.01 % dexamethasone induces skeletal muscle atrophy and impairs the swimming ability of zebrafish through mitochondrial dysfunction and endoplasmic reticulum stress.

Wheat Germ Agglutinin-Modified "Three-in-One" Multifunctional Probe Driven Broad-Spectrum and Flexible Immunochromatographic Diagnosis of viruses With High Sensitivity.

The conventional lateral flow assay (LFA) fails to the demands for the accurate screening of viruses as a result of its low sensitivity of colorimetric signal output and poor universality limited by antibody pairs. Here, a magnetically assisted dual-signal output LFA platform is developed for the ultrasensitive, universal, and flexible detection of viruses. A "three-in-one" multifunctional probe (MAuDQD) is prepared using a 180nm Fe3O4 core to load numerous Au nanoparticles (NPs) and two layers of QDs, which can substantially improve the sensitivity of LFA through coupling with the effects of magnetic enrichment and colorimetric/fluorescent enhancement. Wheat germ agglutinin (WGA)-modified MAuDQD attained the broad-spectrum capture viral membrane proteins and the colorimetric/fluorescent dual-mode detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and monkeypox virus (MPXV) on the LFA strip. In the colorimetric mode, the target viruses detected directly, with the visual sensitivity reaching 0.1-0.5ngmL-1 and the fluorescent mode supported quantitative analysis of SARS-CoV-2/MPXV with limits of detection decreasing to pgmL-1 level. Practicability of the MAuDQD@WGA-LFA is verified through the detection of 33 real clinical samples, showing the proposed assay has a great potential to become a sensitive, accurate, and universal tool for on-site monitoring of viral infections.

Glycosyl Mobile Radical Structures of Folic Acid Receptors Impact the Internalization of Functionalized Folate Amphiphilic Alternating Copolymer in Cancer Cells

Folate receptor alpha (FRα) is a glycosylphosphatidylinositol (GPI) membrane-anchored protein containing three N-glycosylated residues at the N47, N139, and N179 termini. These glycosylation sites have been reported to be crucial for the receptor’s structural integrity and its ability to bind and internalize FA. Here, we investigated the role of FRα glycosylation in the binding and internalization efficacy of FA–DABA–SMA in pancreatic PANC-1 cancer cells. There is a strong association of the FA copolymer with FRα with a Pearson coefficient R-value of 0.7179. PANC-1 cancer cells were pretreated with maackia amurensis lectin II (MAL-2), sambucus Nigra lectin (SNA-1), peanut agglutinin (PNA), and wheat germ agglutinin lectin (WGA) at different doses followed by 20 kDa and 350 kDa FA–DABA–SMA loaded with coumarin 153 (C153). Increasing the dosage of MAL2, SNA-1, PNA, and WGA concomitantly and significantly increased the internalization of C153-loaded FA–DABA–SMA in the cells. The half maximal effective lectin concentrations (EC50) to induce cellular internalization into the cytoplasm of the lectins for MAL-2 were 35.88 µg/mL, 3.051 µg/mL for SNA-1, 7.883 µg/mL for PNA, and 0.898 µg/mL for WGA. Live cell imaging of the internalization of 20 kDa and 350 kDa FA copolymers indicated an aggregation of 350 kDa copolymer with FRα in the cytoplasm. In contrast, the 20 kDa FA copolymer remained in the membrane. The data indicate for the first time that the mobile positions of the glycosyl radical groups and the receptor tilt in generating steric hindrance impacted the individual FRα receptors in the binding and internalization of 350 kDa FA–DABA–SMA in cancer cells.

Synthesis of Multivalent Glycoside-Immobilized Carboxymethyl Cellulose Nanohydrogel Particles with Superadsorption Ability for Lectins.

Carboxymethyl cellulose (CMC) is a water-soluble cellulose derivative that is nontoxic, biocompatible, biodegradable, and nonallergenic. As developing an adsorbent material for carbohydrate-binding proteins is challenging, we aimed to synthesize CMC nanohydrogel particles (CMCGPs) with an extremely high lectin adsorption tendency in this study. CMCGPs were used as the backbone of an adsorption carrier that was synthesized by cross-linking CMC with ethylene glycol diglycidyl ether. A series of glycoside-immobilized CMCGPs were synthesized by binding two types of glycans (LacNAc and lactose) to the polyvalent carboxymethyl groups that are present on the CMCGP surface and act as reaction sites. These immobilized glycosides function as molecular recognition sites. Glycan moieties were incorporated into the CMCGP backbone at degrees of immobilization (DI) ranging from 8.7 to 21.0% by altering the reaction composition. LacNAc-CMCGP (3b) showed a 19.9% DI of LacNAc glycoside to the CMCGP carboxymethyl group; on average, its particle size swelled to 418 nm in phosphate-buffered saline, which is approximately 1.4 times its dry-state size. Analyzing the adsorbent properties of glyco-CMCGPs using a lectin-binding assay showed the high structural specificity of glyco-CMCGPs to lectins. The equilibrium isotherm data was explained by the Langmuir adsorption model. Notably, compound 3b adsorbed 1.95 ± 0.05 μg of wheat germ agglutinin (WGA) lectin per 1.0 μg-dry of 3b particles at an adsorption equilibrium time of a few minutes. Furthermore, solid-state 13C nuclear magnetic resonance analysis showed that WGA lectin retained its natural structure without denaturation after binding to LacNAc-CMCGP. These results were also supported by affinity purification experiments of WGA from raw wheat germ extract using LacNAc-CMCGP, demonstrating that glyco-CMCGP is capable of adsorbing and desorbing lectin while maintaining its biological activity. Thus, multivalent glycoside-immobilized CMCGPs that use woody biomass derivatives as the backbone are expected to be applied as biorefinery materials, which specifically and abundantly adsorb not just plant lectins but also pathogenic viruses and toxin proteins.

Expression of a novel NaD1 recombinant antimicrobial peptide enhances antifungal and insecticidal activities

This study aimed to increase the antifungal and insecticidal activities of NaD1, as an antimicrobial peptides (AMP), by improving its interaction with the fungal cell wall and chitin monomeric units in insect midguts. Hence, the chitin-binding domains (CBDs) of wheat germ agglutinin protein (WGA) were fused to either N- or C-terminus of NaD1 generating transgenic Nicotiana tabacum hairy roots (HRs). Molecular assessments confirmed the integration of NaD1 transgenes, their transcription and production of recombinant peptides in the HR lines. Total protein of (CBD)4-NaD1 and NaD1-(CBD)4 transgenic lines inhibited the growth of Pyricularia oryzae mycelium, suggesting that fusion of CBD to NaD1 can increase NaD1 half-life, leading to higher affinity toward cell wall chitin. Furthermore, feeding the third-instar larvae of Chilo suppressalis with both (CBD)4-NaD1 and NaD1-(CBD)4 extracts exhibited a higher mortality rate. Both NaD1-CBDs caused a significant decrease in trypsin (TRY) and chymotrypsin (CTR) activities in the larvae, while enhancing the activity of antioxidant enzymes CAT, POD, APX, and SOD. Therefore, feeding the larvae by total extract of NaD1-(CBD)4 and (CBD)4-NaD1 HR lines probably increased affinity to midgut chitin in C. suppressalis, enhancing insecticidal activities. Overall, the results indicate that recombinant peptides are effective in enhancing fungal and insect resistance.

Epidermal distribution of tetrodotoxin-rich cells in newly hatched larvae of Takifugu spp.

Pufferfish of the genus Takifugu possess tetrodotoxin (TTX), known as “pufferfish toxin” and it is believed that pufferfish eggs and newly hatched larvae utilize TTX as a defensive substance against predators. However, the mechanism for the placement of TTX to specific cells on the larval body surface during the developmental process remains unknown. In this study, we clarify the distribution and characteristics of TTX-rich cells. We performed whole-mount immunohistochemistry (IHC) using anti-TTX monoclonal antibody on larvae of two pufferfish species, Takifugu rubripes and Takifugu alboplumbeus, just after hatching. This allowed observation of the TTX location and compared it with those of wheat germ agglutinin (WGA)-positive (periodic acid-Schiff (PAS)-positive) cells for mucous cells and IHC using anti-Na+/K+-ATPase (NKA) monoclonal antibody for ionocytes. As a result, uniformly scattered localization of TTX-rich cells was commonly observed in the epidermis of the larvae of the two Takifugu species. TTX-rich cells were WGA-negative (PAS-negative) and structurally distinct from NKA-positive cells, suggesting that TTX-rich cells are unreported small cells unique to pufferfish skin, but not mucous cells nor ionocytes.

Novel Fluorescence-Based Methods to Determine Infarct and Scar Size in Murine Models of Reperfused Myocardial Infarction.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson's (r = 0.9920) and Gomori's Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

Role of chitin synthases CHS1 and CHS2 in biosynthesis of the cyst wall of Cryptocaryon irritans

Cryptocaryon irritans, a protozoan parasite that infects marine fish, is characterized by a complex life cycle that includes a cyst-forming reproductive phase. However, the composition of the cyst wall and mechanism of its formation remain unclear. In this study, we identified chitin as a key component of the cyst wall using calcofluor white and wheat germ agglutinin, with Fourier-transform infrared spectroscopy confirming its β-form structure. Two chitin synthase genes, CHS1 and CHS2, were identified as being expressed throughout the life cycle and show close phylogenetic relationships with chitin synthase from ciliates. Incubation with specific anti-CHS1 and -CHS2 antibodies significantly reduced both the thickness and chitin content of the cyst wall, highlighting the critical role of these enzymes in chitin biosynthesis. Treatment with benzoylureas, which inhibit chitin synthesis, caused thinning of the cyst wall and downregulation of CHS gene expression, resulting in an 84 % reduction in the hatching rate after treatment with 0.01 mM CuSO4 compared with control tomonts. Western blot analysis demonstrated that recombinant CHS proteins are immunogenic, and tomonts from CHS-immunized grouper exhibited reduced size. These findings bridge a crucial knowledge gap in understanding of the C. irritans cyst wall and highlight promising targets for infection prevention and control strategies.

Characteristics of lectin receptors on erythrocyte membranes of patients with glioblastoma

Aim. This work is devoted to the search the abnormalities of the receptor-lectin reactions associated with glioblastoma for diagnostic purposes. Methods. Red blood cell (RBC) specimens were taken from 22 persons distributed into the two cohorts: donors without an oncological diagnosis; patients with glioblastoma (GB). Panel of 9 commercial lectins (LECTINOTEST, Lviv, Ukraine) was used: ConA, WGA, PHA-P; PHA-E; PHA-L, SNA, VAA, STA, and LABA. Also the original lectin from persimmon sepals (named PSL) was used. It was obtained by isoelectric focusing and ammonium sulfate fractionation. Intensity of lectin-receptor interactions was determined in reaction of hemagglutination. Results. Тwo lectins showed some directed divergence in patients from donors. For wheat germ agglutinin (WGA) сhanges in the intensity of lectin-receptor binding were downward. In the case of persimmon L (PSL) the alternative result was observed: the intensity of hemagglutination showed a trend to exceed the level of control. Conclusions. Some individual features in the reaction of erythrocytes with a set of lectins are observed in all examined persons. For two studied lectins, namely WGA and PSL, unidirectional deviations were observed in the reaction of erythrocytes of patients with GB compared to individuals without an oncological diagnosis.

Assembly and evaluation of a confocal microscopy image analysis pipeline useful in revealing the secrets of plant-fungal interactions

The ability of laser scanning confocal microscopy to generate high-contrast 2D and 3D images has become essential in studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins has several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging pipeline that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This pipeline relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability, allowing the different stains to penetrate and effectively bind to their targets, resulting in a consistent visualization of cellular structures. To expand the utility of this pipeline, we used the staining techniques in conjunction with machine learning to analyze fungal biomass through volume analysis, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This pipeline is simple to use, robust, consistent across host and fungal species and can be applied to most plant-fungal interactions. Therefore, this pipeline can be used to characterize model systems as well as non-model interactions where transformation is not routine.

Wheat Germ Agglutinin (WGA): Its Nature, Biological Role, Significance in Human Nutrition, and Possibility to Be Used as Marker of Whole-Grain Status in Wheat-Based Foods.

The growing scientific evidence on the health benefits of whole-grain food consumption has promoted the manufacturing of a great number of products differing in quality and content of whole-grain components. This is particularly true for commercial wheat-based products where it is not always clear how much whole wheat is present considering that in many cases, they are manufactured from reconstituted mill streams and that there is not a standardised globally accepted definition and metrics to objectively evaluate whole-grain status. Attempts have been made to assess the level of "wholegraininess" in wheat products by measuring specific constituents that correlate with different wheat tissues, especially those that are expected to be found in a true whole-grain wheat product. Wheat germ agglutinin (WGA), a small lectin protein present exclusively in the wheat-germ tissues, has been indicated by several scientists as one of these constituents and after founding that its level changes depending on the amount of germ found in a wheat flour, it has been indicated as a biomarker of whole-grain status for wheat products. In this review, the biochemistry of WGA, its methods of detection, and current knowledge on its possibility to be practically utilized as a reliable marker are critically discussed.