Linear ß-1,2-glucans trigger immune hallmarks and enhance disease resistance in plants.

Immune responses in plants are triggered by molecular patterns or elicitors, recognized by plant pattern recognition receptors. Such molecular patterns are consequence of host-pathogen interactions and the response cascade activated after their perception is known as pattern-triggered immunity (PTI). Glucans have emerged as key players in PTI, but the ability of certain glucans to stimulate defensive responses in plants remains understudied. This work focused on identifying novel glucan oligosaccharides as molecular patterns. The ability of various microorganism-derived glucans to prompt PTI responses was tested, revealing that specific microbial-derived molecules, such as short linear ß-1,2-glucans, trigger this response in plants by increasing the production of reactive oxygen species (ROS), MAP kinase phosphorylation, and differential expression of defence-related genes in Arabidopsis thaliana. Pretreatments with ß-1,2-glucan trisaccharide (B2G3) improved Arabidopsis defence against bacterial and fungal infections in a hypersusceptible genotype. The knowledge generated was then transferred to the monocotyledonous model species maize and wheat, confirming that these plants also respond to ß-1,2-glucans, with increased ROS production and improved protection against fungal infections following B2G3 pretreatments. In summary, as with other ß-glucans, plants perceive ß-1,2-glucans as warning signals and stimulate defence responses against phytopathogens.

USP7 Promotes TGF-ß1 Signaling by De-Ubiquitinating Smad2/Smad3 in Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a long-term, progressive, and irreversible pulmonary interstitial disease. The activation of Smad family member 2 (Smad2) and Smad3 transcription factors by transforming growth factor ß-1 (TGF-ß1) is a critical event in the pathogenesis of IPF. However, there is still a lack of understanding regarding the molecular mechanisms governing Smad2 and Smad3 proteins. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a vital role in regulating protein stability within cells. However, its regulation of the TGF-ß signaling pathway and its significance in IPF remain undiscovered. This study aims to clarify the function of USP7 in the TGF-ß signaling pathway, while simultaneously exploring the specific molecular mechanisms involved. Additionally, this study seeks to evaluate the therapeutic potential of targeted USP7 inhibitors in IPF, thereby providing novel insights for the diagnosis and management of IPF. METHODS: We first detected the expression of USP7 in lung tissues of mice with Bleomycin (BLM)-induced pulmonary fibrosis and in Beas-2B cells treated with or without TGF-ß1 through Western blot analysis. Subsequently, we explored the influence of USP7 on fibrotic processes and the TGF-ß1 signaling pathway, utilizing in vitro and in vivo studies. Finally, we assessed the effectiveness of USP7-specific inhibitors in an IPF murine model. RESULTS: In the present study, USP7 was found to de-ubiquitinate Smad2 and Smad3, consequently increasing their stability and promoting the TGF-ß1-induced production of profibrotic proteins including α-smooth muscle actin (α-SMA) and fibronectin 1 (FN-1). Inhibition or knockdown of USP7 resulted in decreased levels of Smad2 and Smad3 proteins, leading to reduced expression of FN-1, Collagen Type I Alpha 1 Chain (Col1A1), and α-SMA induced by TGF-ß1 in human pulmonary epithelial cells. These findings demonstrate that overexpression of USP7 reduces Smad2/3 ubiquitination, whereas inhibition or knockdown of USP7 enhances their ubiquitination. USP7 is abundantly expressed in IPF lungs. The expressions of USP7, Smad2, and Smad3 were upregulated in bleomycin-induced lung injury. The USP7 inhibitor P22077 reduced the expression of FN-1 and type I collagen as well as Smad2/3 and collagen deposition in lung tissue in a model of pulmonary fibrosis induced by bleomycin. CONCLUSIONS: This study demonstrates that USP7 promotes TGF-ß1 signaling by stabilizing Smad2 and Smad3. The contribution of USP7 to the progression of IPF indicates it may be a viable treatment target.

[Expression of transferrin receptor 1 and ß1-integrins correlates with estrogen receptor status and immune infiltration in breast cancer]. / Ekspressiya transferrinovykh retseptorov 1 i ß1-integrinov korreliruet so statusom retseptorov estrogenov i immunnoi infil'tratsiei pri rake molochnoi zhelezy.

Cancer cells can aberrantly express various markers, including transferrin receptor 1 (CD71) and ß1-integrin molecules. Their role in invasion, migration and metastasis has been demonstrated. Determination of their expression in breast cancer (BC) may be an important point to characterize the clinical course of the tumor and prognosis of the disease. OBJECTIVE: To study of transferrin receptor 1 (CD71) expression by primary breast cancer cells in correlation with tumor cell phenotype. MATERIAL AND METHODS: Determination of BC phenotype: immunohistochemical staining method (immunofluorescence). Antibodies to ER (estrogen receptors), KL-1 (pancytokeratin), CD71 (transferrin receptor), CD29 (ß1-integrins). CD45, CD3, CD4, CD8, CD20 infiltration was also evaluated. ZEISS microscope (AXIOSKOP; Germany), method of G.J. Hammerling et al. Statistical processing: IBM-SPSS Statistics v.21. RESULTS: 63% of BC cases had CD71+ phenotype. CD71-mosaic tumors were observed in 14.4%. ß1-integrin expression was monomorphic in 51.6% of cases and mosaic in 38.7%. 85% of ER-positive tumors were CD71-positive with a monomorphic type of reaction; p=0.014. Among ER-negative tumors, CD71-negative reactions were 2-fold more frequent and the monomorphic type was less frequent. ER-positive tumors were CD29-positive in 73%; p=0.031. 45.5% of ER+ tumors were CD29-monomorphic. Among ER-negative tumors, the frequency of CD29-monomorphic tumors was 55%. Significant infiltration by CD3+ cells was predominant in CD71-positive tumors; p=0.016. In the CD29-monomorphic phenotype, CD45+ infiltration was 31.3%, and in the mosaic phenotype, 67.1%. CONCLUSION: BC aberrantly expresses transferrin receptors, ß1-integrins. CD71 expression is associated with ER expression. ER-positive tumors are often monomorphic for CD71. Prominent CD3+ infiltration was present in CD71+ tumors. Expression of ß1-integrins correlated with ER+ status and weak immune infiltration.

Inhibition and reversal of a TGF-ß1 induced myofibroblast phenotype by adipose tissue-derived paracrine factors.

BACKGROUND: Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation. METHODS: The transforming growth factor-ß1 (TGF-ß1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts. RESULTS: Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-ß1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue. CONCLUSIONS: Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism.

Combining experiments and bioinformatics to identify transforming growth factor-ß1 as a key regulator in angiotensin II-induced trophoblast senescence.

INTRODUCTION: Accelerated senescence of trophoblast may cause several diverse pregnancy outcomes; however, the cause of accelerated trophoblast senescence remains unclear. The renin-angiotensin system (RAS) is closely related to organ senescence. Therefore, in the present study, we hypothesized that angiotensin (Ang)II, one of the most important RAS family members, accelerates trophoblast senescence through the transforming growth factor ß-1 (TGF-ß1) pathway. METHODS: AngII and Ang1-7 were used to stimulate pregnant rats. AngII and its inhibitor olmesartan were used to stimulate trophoblast. Thereafter, senescence levels were measured. Furthermore, we used AngII to stimulate trophoblast and utilized RNA-sequencing (RNAseq) to analyze the expression of differentially expressed genes (DEGs). After identifying the overlapping genes by comparing the DEGs and senescence-related genes, we employed CytoHubba software to calculate the top five hub genes and selected TGF-ß1 as the target gene. We transfected the AngII-stimulated trophoblast with TGF-ß1 small interfering RNA (siRNA) and measured the senescence levels. RESULTS: Senescence markers were upregulated in the AngII group compared with that in the control group. Furthermore, following AngII stimulation and RNAseq measurement, we identified 607 DEGs and 13 overlapping genes. The top five hub genes were as follows: PLAU, PTGS2, PDGF-ß, TGF-ß1, and FOXO3. Upon knockdown of TGF-ß1 expression in AngII-stimulated trophoblast using TGF-ß1 siRNA, we observed a downregulation of p53 and p62 mRNA expression. DISCUSSION: AngII accelerates trophoblast senescence through the TGF-ß1 pathway.

Role of proteoglycan synthesis genes in osteosarcoma stem cells.

Osteosarcoma stem cells (OSCs) contribute to the pathogenesis of osteosarcoma (OS), which is the most common malignant primary bone tumor. The significance and underlying mechanisms of action of proteoglycans (PGs) and glycosaminoglycans (GAGs) in OSC phenotypes and OS malignancy are largely unknown. This study aimed to investigate the role of PG/GAG biosynthesis and the corresponding candidate genes in OSCs and poor clinical outcomes in OS using scRNA-seq and bulk RNA-seq datasets of clinical OS specimens, accompanied by biological validation by in vitro genetic and pharmacological analyses. The expression of ß-1,3-glucuronyltransferase 3 (B3GAT3), one of the genes responsible for the biosynthesis of the common core tetrasaccharide linker region of PGs, was significantly upregulated in both OSC populations and OS tissues and was associated with poor survival in patients with OS with high stem cell properties. Moreover, the genetic inactivation of B3GAT3 by RNA interference and pharmacological inhibition of PG biosynthesis abrogated the self-renewal potential of OSCs. Collectively, these findings suggest a pivotal role for B3GAT3 and PG/GAG biosynthesis in the regulation of OSC phenotypes and OS malignancy, thereby providing a potential target for OSC-directed therapy.

Molecular cloning, characterisation and molecular modelling of two novel T-synthases from mollusc origin.

The glycoprotein-N-acetylgalactosamine ß1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is the core 1 O-glycan structure. This enzyme transfers galactose from UDP-Gal to GalNAc-Ser/Thr. The T-antigen has significant functions in animal development, immune response, and recognition processes. Molluscs are a successful group of animals that inhabit various environments, such as freshwater, marine, and terrestrial habitats. They serve important roles in ecosystems as filter feeders and decomposers but can also be pests in agriculture and intermediate hosts for human and cattle parasites. The identification and characterization of novel carbohydrate active enzymes, such as T-synthase, can aid in the understanding of molluscan glycosylation abilities and their adaptation and survival abilities. Here, the T-synthase enzymes from the snail Pomacea canaliculata and the oyster Crassostrea gigas are identified, cloned, expressed, and characterized, with a focus on structural elucidation. The synthesized enzymes display core 1 ß1,3-galactosyltransferase activity using pNP-α-GalNAc as substrate and exhibit similar biochemical parameters as previously characterised T-synthases from other species. While the enzyme from C. gigas shares the same structural parameters with the other enzymes characterised so far, the T-synthase from P. canaliculata lacks the consensus sequence CCSD, which was previously considered indispensable.

Exploring a novel ß-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides.

The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses ß-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming ß-1,3 glucan acceptor, making a ß-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several ß-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-ß-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-ß-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.

Experimental Study on the Effect of Caffeine Hydrogel on the Expression of TGF -ß1, α-SMA and Collagen in Hypertrophic Scar of Rabbit Ears.

This study evaluated the effects of topical use of caffeine hydrogel on hypertrophic scar in a rabbit ear wound model. Nine rabbits were randomly divided into three groups: control group, caffeine hydrogel group, and matrix group. Punched defects were established on each rabbit's ear which resulted in a hypertrophic scar. When the wound epithelialization and scar hyperplasia could be seen, control group did not do any treatment, while caffeine hydrogel group and matrix group were treated with caffeine hydrogel and hydrogel matrix, respectively. After 3 weeks of administration, the general morphological changes of scar were observed, and the scar tissue of rabbit ears was stained with HE and Masson. The relative expressions of TGF ß-1, α-SMA, type I collagen, and type III collagen in scar tissue were detected by Western blot. In all three groups, findings showed that caffeine hydrogel can inhibit scar growth by reducing the expression of TGF ß-1, reducing the proliferation of fibroblasts, improving collagen arrangement and reducing collagen deposition. The overall study shows efficacy and mechanism of caffeine. It concluded that caffeine could be an effective therapeutic agent for hypertrophicscars.

Biochemical and structural characterization of a glucan synthase GFGLS2 from edible fungus Grifola frondosa to synthesize ß-1, 3-glucan.

BACKGROUND: Grifola frondosa is a Basidiomycete fungus belonging to the family of Grifolaceae and the order of Polyporales. ß-Glucans are the main polymers in G. frondosa, playing a crucial role in the physiology and representing the healthy benefits for humans. The membrane-integrated ß-1, 3-glucan synthase (GLS) is responsible for glucan synthesis, cell wall assembly, differentiation and growth of the edible fungi. However, the structural/catalytic characteristics and mechanisms of ß-1, 3-glucan synthases in G. frondosa are still unknown due to their extremely complex structures with multi-transmembranes and large molecular masses. RESULTS: Herein, a ß-1, 3-glucan synthase (GFGLS2) was purified and identified from the cultured mycelia with a specific activity of 60.01 pmol min-1 µg-1 for the first time. The GFGLS2 showed a strict specificity to UDP-glucose with a Vmax value of 1.29 ± 0.04 µM min-1 at pH 7.0 and synthesized ß-1, 3-glucan with a maximum degree of polymerization (DP) of 62. Sequence Similarity Network (SSN) analysis revealed that GFGLS2 has a close relationship with others in Ganoderma sinense, Trametes coccinea, Polyporus brumalis, and Trametes pubescens. With the assistance of 3D structure modelling by AlphaFold 2, molecular docking and molecular dynamics simulations, the central hydrophilic domain (Class III) in GFGLS2 was the main active sites through binding the substrate UDP-glucose to 11 amino acid residues via hydrogen bonds, π-stacking and salt bridges. CONCLUSIONS: The biochemical, 3D structural characterization and potential catalytic mechanism of a membrane-bound ß-1, 3-glucan synthase GFGLS2 from cultured mycelia of G. frondosa were well investigated and would provide a reasonable full picture of ß-1, 3-glucan synthesis in fungi.

The salt-activated CBF1/CBF2/CBF3-GALS1 module fine-tunes galactan-induced salt hypersensitivity in Arabidopsis.

Plant growth and development are significantly hampered in saline environments, limiting agricultural productivity. Thus, it is crucial to unravel the mechanism underlying plant responses to salt stress. ß-1,4-Galactan (galactan), which forms the side chains of pectic rhamnogalacturonan I, enhances plant sensitivity to high-salt stress. Galactan is synthesized by GALACTAN SYNTHASE1 (GALS1). We previously showed that NaCl relieves the direct suppression of GALS1 transcription by the transcription factors BPC1 and BPC2 to induce the excess accumulation of galactan in Arabidopsis (Arabidopsis thaliana). However, how plants adapt to this unfavorable environment remains unclear. Here, we determined that the transcription factors CBF1, CBF2, and CBF3 directly interact with the GALS1 promoter and repress its expression, leading to reduced galactan accumulation and enhanced salt tolerance. Salt stress enhances the binding of CBF1/CBF2/CBF3 to the GALS1 promoter by inducing CBF1/CBF2/CBF3 transcription and accumulation. Genetic analysis suggested that CBF1/CBF2/CBF3 function upstream of GALS1 to modulate salt-induced galactan biosynthesis and the salt response. CBF1/CBF2/CBF3 and BPC1/BPC2 function in parallel to regulate GALS1 expression, thereby modulating the salt response. Our results reveal a mechanism in which salt-activated CBF1/CBF2/CBF3 inhibit BPC1/BPC2-regulated GALS1 expression to alleviate galactan-induced salt hypersensitivity, providing an activation/deactivation fine-tune mechanism for dynamic regulation of GALS1 expression under salt stress in Arabidopsis.

Evaluation of the effects of sinapic acid and ellagic acid on sciatic nerve in experimental diabetic rats by immunohistochemical and stereological methods.

In our study, we aimed to examine the effects of sinapic acid and ellagic acid on neuropathy caused by diabetes in peripheral nerves. Fifty-six adult Wistar Albino rats Control, Diabetes, Diabetes+Sinapic Acid, Diabetes+Ellagic Acid, Diabetes+Sinapic Acid+Ellagic Acid, Sinapic Acid, Ellagic Acid and as Sinapic Acid+Ellagic Acid, they were randomly divided into eight groups(n:7). A single dose of 50 mg/kg streptozotocin(STZ) was administered intraperitoneally to the groups to be diagnosed with diabetes. Diabetes was accepted as blood glucose value of 250 mg/dL and above. Streptozotocin was given to the diabetes groups, 20 mg/kg/day intragastric Sinapic acid to the Sinapic acid groups, 50 mg/kg/day intragastric Ellagic acid to the Ellagic acid groups for 28 days. At the end of the experiment, 0.5 cm of the right sciatic nerve was removed. It was fixed in 10% formaldehyde. After histological follow-up, it was embedded in paraffin, 5 µm thick sections were taken. Immunohistochemical staining with Fibrinogen alpha, Laminin ß-1 and Collagen IV antibodies and stereological evaluation was performed by Physical Dissector Combination method. Collagen IV was used in control, diabetes and treatment groups showed similar immunostaining. Fibrinogen alpha was observed to be increased in the vessel wall in the diabetes group, while the uptake was minimal in the control and treatment groups. While Laminin ß-1 was increased in the diabetes group compared to the control group, immunostaining was observed in the treatment groups similar to the control group. It was observed that the total nerve area diabetes group decreased significantly compared to the control group, and the treatment groups, except for D+EA group were similar to the control group, but there was no statistically significant difference. The axon numbers in the diabetes group decreased significantly compared to the control group, and the treatment groups were similar to the control group, and there was no statistically significant difference (P > 0.05). It was determined that Sinapic Acid and Ellagic acid had positive effects on the nervous tissue in diabetic neuropathy.

Myxobacterial Outer Membrane ß-1,6-Glucanase Induced the Cell Death of Fusarium oxysporum by Destroying the Cell Wall Integrity.

The ß-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and ß-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The ß-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 µg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of ß-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to ß-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. IMPORTANCE Phytopathogenic fungi are the most devastating plant pathogens in agriculture, causing enormous economic losses to global crop production. Biocontrol agents have been promoted as replacements to synthetic chemical pesticides for sustainable agriculture development. Cell wall-degrading enzymes (CWDEs), including chitinases and ß-1,3-glucanases, have been considered as important armaments to damage the cell wall. Here, we found that F. oxysporum f. sp. cucumerinum is more sensitive to ß-1,6-glucanase GluM treatment (0.08 µg/mL) than ß-1,3-glucanase Zymolyase (1.0 mg/mL). The HOG pathway was activated in F. oxysporum f. sp. cucumerinum cells after GluM treatment, and the intracellular glycerol content was significantly increased. Moreover, the decomposition of F. oxysporum f. sp. cucumerinum cell wall by GluM induced the burst of intracellular ROS and apoptosis, which eventually leads to cell death. Therefore, we suggest that the ß-1,6-glucan of the fungal cell wall may be a better antifungal target compared to the ß-1,3-glucan.

Cellobiose elicits immunity in lettuce conferring resistance to Botrytis cinerea.

Cellobiose is the primary product of cellulose hydrolysis and is expected to function as a type of pathogen/damage-associated molecular pattern in evoking plant innate immunity. In this study, cellobiose was demonstrated to be a positive regulator in the immune response of lettuce, but halted autoimmunity when lettuce was exposed to concentrations of cellobiose >60 mg l-1. When lettuce plants were infected by Botrytis cinerea, cellobiose endowed plants with enhanced pre-invasion resistance by activating high ß-1,3-glucanase and antioxidative enzyme activities at the initial stage of pathogen infection. Cellobiose-activated core regulatory factors such as EDS1, PTI6, and WRKY70, as well as salicylic acid signaling, played an indispensable role in modulating plant growth-defense trade-offs. Transcriptomics data further suggested that the cellobiose-activated plant-pathogen pathways are involved in microbe/pathogen-associated molecular pattern-triggered immune responses. Genes encoding receptor-like kinases, transcription factors, and redox homeostasis, phytohormone signal transduction, and pathogenesis-related proteins were also up- or down-regulated by cellobiose. Taken together, the findings of this study demonstrated that cellobiose serves as an elicitor to directly activate disease-resistance-related cellular functions. In addition, multiple genes have been identified as potential modulators of the cellobiose-induced immune response, which could aid understanding of underlying molecular events.

The humidity level matters during the desiccation of Norway spruce somatic embryos.

In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes ß-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some ß-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation.

A Potent Micron Neoantigen Tumor Vaccine GP-Neoantigen Induces Robust Antitumor Activity in Multiple Tumor Models.

Therapeutic tumor neoantigen vaccines have been widely studied given their good safety profile and ability to avoid central thymic tolerance. However, targeting antigen-presenting cells (APCs) and inducing robust neoantigen-specific cellular immunity remain challenges. Here, a safe and broad-spectrum neoantigen vaccine delivery system is proposed (GP-Neoantigen) based on ß-1,3-glucan particles (GPs) derived from Saccharomyces cerevisiae and coupling peptide antigens with GPs through convenient click chemistry. The prepared system has a highly uniform particle size and high APC targeting specificity. In mice, the vaccine system induced a robust specific CD8+ T cell immune response and humoral immune response against various conjugated peptide antigens and showed strong tumor growth inhibitory activity in EG7·OVA lymphoma, B16F10 melanoma, 4T1 breast cancer, and CT26 colon cancer models. The combination of the toll-like receptors (TLRs) agonist PolyI:C and CpG 2395 further enhanced the antitumor response of the particle system, achieving complete tumor clearance in multiple mouse models and inducing long-term rejection of reinoculated tumors. These results provide the broad possibility for its further clinical promotion and personalized vaccine treatment.

High iron-mediated increased oral fungal burden, oral-to-gut transmission, and changes to pathogenicity of Candida albicans in oropharyngeal candidiasis.

BACKGROUND: Iron affects the diversity of the oral microbial landscape. Laboratory-strain CAI4 of Candida albicans that causes oropharyngeal candidiasis (OPC) exhibits iron-induced changes to the cell wall, impacting phagocytosis (by macrophages) and susceptibility of fungal cells to cell wall-perturbing antifungals, in vitro. AIM: To understand the effect of iron on the CAI4-strain, wild type (WT) SC5314-strain, and oral isolates of C. albicans. METHODS: An immunosuppressed murine model of OPC was used to assess the effect of iron on oral-to-gut infection and antifungal susceptibility of the CAI4-strain. In vitro antifungal susceptibility, cell wall analysis, and phagocytic assays were performed under low and high iron, for the SC5314-strain and oral isolates. RESULTS: High iron enhanced oral and gut fungal levels for the CAI4-strain in mice; CAI4 cells from low iron mice were more susceptible to antifungals. The SC5314-strain and oral isolates showed enhanced antifungal-resistance towards most antifungals tested, under high iron. Iron-mediated cell wall changes and phagocytic response in the SC5315-strain were similar to CAI4; oral isolates showed a variable response. CONCLUSION: Host iron can potentially alter infection severity and dissemination, efficacy of antifungal treatment, and host immune response during OPC. Clinical isolates showed most of these effects of iron, despite exhibiting a varied cell wall composition-change response to iron.

DNMTs Are Involved in TGF-ß1-Induced Epithelial-Mesenchymal Transitions in Airway Epithelial Cells.

Chronic rhinosinusitis (CRS) pathogenesis is closely related to tissue remodeling, including epithelial-mesenchymal transition (EMT). Epigenetic mechanisms play key roles in EMT. DNA methylation, mediated by DNA methyltransferases (DNMTs), is an epigenetic marker that is critical to EMT. The goal of this study was to determine whether DNMTs were involved in TGF-ß1-induced EMT and elucidate the underlying mechanisms in nasal epithelial cells and air-liquid interface cultures. Global DNA methylation and DNMT activity were quantified. DNMT expression was measured using real-time PCR (qRT-PCR) in human CRS tissues. mRNA and protein levels of DNMTs, E-cadherin, vimentin, α-SMA, and fibronectin were determined using RT-PCR and Western blotting, respectively. DNMT1, DNMT3A, and DNMT3B gene expression were knocked down using siRNA transfection. MAPK phosphorylation and EMT-related transcription factor levels were determined using Western blotting. Signaling pathways were analyzed using specific inhibitors of MAPK. We demonstrated these data in primary nasal epithelial cells and air-liquid interface cultures. Global DNA methylation, DNMT activity, and DNMT expression increased in CRS tissues. DNMT expression was positively correlated with Lund-McKay CT scores. TGF-ß1 dose-dependently induced DNMT expression. Further, 5-Aza inhibited TGF-ß1-induced DNMT, Snail, and Slug expression related to EMT, as well as p38 and JNK phosphorylation in A549 cells and TGF-ß1-induced DNMT expression and EMT in primary nasal epithelial cells and air-liquid interface cultures. TGF-ß1-induced DNMT expression leads to DNA methylation and EMT via p38, JNK, Snail, and Slug signaling pathways. Inhibition of DNMT suppressed the EMT process and therefore is potentially a CRS therapeutic strategy.

A Simple Technique for Direct Immobilization of Target Enzymes from Cell Lysates Based on the SpyTag/SpyCatcher Spontaneous Reaction.

Many of the current methods for enzyme purification and immobilization suffer from several drawbacks, such as requiring tedious multistep procedures or long preparation, and being environmentally unfriendly, due to the chemicals and conditions involved. Thus, a simple technique for direct purification and immobilization of target enzymes from cell lysates was proposed. The elastin-like polypeptides (ELPs)-SpyCatcher chimera could mediate the formation of silica carriers within seconds and the target enzymes were then covalently immobilized on silica carriers via SpyCatcher/SpyTag spontaneous reaction. These tailor-made carriers were easily prepared, with precisely controlled morphology and size, as well as none-consuming surface modification needed, which could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification.