Journal of advanced research最新文献

Introduction: Heat stress poses a severe threat to the growth and production of soybean (Glycine max). Brassinosteroids (BRs) actively participate in plant responses to abiotic stresses, however, the role of BR signaling pathway genes in response to heat stress in soybean remains poorly understood.

Objectives: In this study, we investigate the regulatory mechanisms of GmBSK1 and GmBES1.5 in response to heat stress and the physiological characteristics and yield performance under heat stress conditions.

Methods: Transgenic technology and CRISPR/Cas9 technology were used to generated GmBSK1-OE, GmBES1.5-OE and gmbsk1 transgenic soybean plants, and transcriptome analysis, LUC activity assay and EMSA assay were carried out to elucidate the potential molecular mechanism underlying GmBSK1-GmBES1.5-mediated heat stress tolerance in soybean.

Results: CRISPR/Cas9-generated gmbsk1 knockout mutants exhibited increased sensitivity to heat stress due to a reduction in their ability to scavenge reactive oxygen species (ROS). The expression of GmBES1.5 was up-regulated in GmBSK1-OE plants under heat stress conditions, and it directly binds to the E-box motif present in the promoters of abiotic stress-related genes, thereby enhancing heat stress tolerance in soybean plants. Furthermore, we identified an interaction between GmGSK1 and GmBES1.5, while GmGSK1 inhibits the transcriptional activity of GmBES1.5. Interestingly, the interaction between GmBSK1 and GmGSK1 promotes the localization of GmGSK1 to the plasma membrane and releases the transcriptional activity of GmBES1.5.

Conclusion: Our findings suggest that both GmBSK1 and GmBES1.5 play crucial roles in conferring heat stress tolerance, highlighting a potential strategy for breeding heat-tolerant soybean crops involving the regulatory module consisting of GmBSK1-GmGSK1-GmBES1.5.

Introduction: Mechanical stresses incurred during post-harvest fruit storage and transportation profoundly impact decay and losses. Currently, the monitoring of mechanical forces is primarily focused on vibrational forces experienced by containers and vehicles and impact forces affecting containers. However, the detection of compressive forces both among interior fruit and between fruit and packaging surfaces remains deficient. Hence, conformable materials capable of sensing compressive stresses are necessary.

Objectives: In the present study, a triple-network-reinforced PSA/LiCl/CCN@AgNP conductive hydrogel was synthesized for compression force detection on fruit surfaces based on changes in intrinsic impedance under mechanical loading.

Methods: The conductive hydrogel was characterized in terms of its adhesion, mechanics, frost resistance, water retention, conductivity, mechanical force-sensing properties, and feasibility for monitoring mechanical forces. Then, a portable complex impedance recorder was developed to interface with the conductive hydrogel and its mechanical force sensing ability was evaluated.

Results: Beyond its inherent conductivity, the hydrogel exhibited notable pressure sensitivity within the strain range of 1 % to 80 %. The conductive hydrogel also demonstrated a commendable adhesion property, favorable tensile property (580 % elongation at break), substantial compressive strength and durability, and a long-term water retention capability. After exposure to -20 °C for 96 h, the hydrogel maintained its mechanical strength, affirming its anti-freezing property. In addition, a portable complex impedance recorder with sustained signal measurement stability was developed to quantitatively acquire the hydrogel resistance changes in response to compression forces. Finally, the effectiveness of the conductive hydrogel for sensing compression force on the surface of apple fruits was validated.

Conclusion: The conductive hydrogel holds promise for applications in smart packaging, wherein it can detect crucial mechanical stress on fruit, convert it into electrical signals, and further transmit these signals to the cloud, thereby enabling the real-time sensing of mechanical forces experienced by fruits and enhancing post-harvest fruit loss management.

Introduction: Plant bacterial diseases take an incalculable toll on global food security. The indiscriminate use of chemical synthetic pesticide not only facilitates pathogen resistance of pathogenic bacteria, but also poses a major threat to human health and environmental protection. Therefore, it is of great economic value and scientific significance to develop a new antibacterial drug with environmental friendliness and unique mechanism of action.

Objectives: To design and synthesize formononetin derivatives based on natural products, evaluate their in vitro and in vivo antibacterial activities and elucidate the mechanisms involved.

Methods: The synthesis was carried out by classical active group splicing method. The antibacterial activities were evaluated using turbidimetry and pot experiments. The antibacterial mechanism was further investigated using scanning electron microscopy (SEM), virulence factors, defense enzymes activities, proteomics and metabolomics.

Results: 40 formononetin derivatives containing benzyl piperidine were designed and synthesized. The antibacterial results demonstrated that H32 exhibited the most potent inhibitory effect against Xanthomonas oryzae pv. Oryzae (Xoo) with the EC₅₀ of 0.07 μg/mL, while H6 displayed the highest inhibitory activity against Xanthomonas axonopodis pv. Citri (Xac) with the EC₅₀ of 0.24 μg/mL. Furthermore, the control efficacy of H32 against rice bacterial leaf blight (BLB) and H6 against citrus canker (CC) was validated through pot experiments. SEM, virulence factors and host enzyme activities assay indicated that H32 could not only reduce the virulence of Xoo, but also activate the activities of defense enzymes and improve the disease resistance of host plants. The proteomics and metabolomics analysis demonstrated that H32 could inhibit the synthesis of branched-chain amino acids, make Xoo cells in a starvation state, inhibit its proliferation, weaken its virulence and reduce its colonization and infection of host cells.

Conclusion: Formononetin derivatives containing benzyl piperidine could be used as potentially effective inhibitors against Xanthomonas spp.

Introduction: Host-microbe interactions are important to human health and ecosystems globally, so elucidating the complex host-microbe interactions and associated protein expressions drives the need to develop sensitive and accurate biochemical techniques. Current proteomics techniques reveal information from the point of view of either the host or microbe, but do not provide data on the corresponding partner. Moreover, it remains challenging to simultaneously study host-microbe proteomes that reflect the direct competition between host and microbe. This raises the need to develop a dual-species proteomics method for host-microbe interactions.

Objectives: We aim to establish a forward + reverse Stable Isotope Labeling with Amino acids in Cell culture (SILAC) proteomics approach to simultaneously label and quantify newly-expressed proteins of host and microbe without physical isolation, for investigating mechanisms in direct host-microbe interactions.

Methods: Using Caenorhabditis elegans-Pseudomonas aeruginosa infection model as proof-of-concept, we employed SILAC proteomics and molecular pathway analysis to characterize the differentially-expressed microbial and host proteins. We then used molecular docking and chemical characterization to identify chemical inhibitors that intercept host-microbe interactions and eliminate microbial infection.

Results: Based on our proteomics results, we studied the iron competition between pathogen iron scavenger and host iron uptake protein, where P. aeruginosa upregulated pyoverdine synthesis protein (PvdA) (fold-change of 5.2313) and secreted pyoverdine, and C. elegans expressed ferritin (FTN-2) (fold-change of 3.4057). Targeted intervention of iron competition was achieved using Galangin, a ginger-derived phytochemical that inhibited pyoverdine production and biofilm formation in P. aeruginosa. The Galangin-ciprofloxacin combinatorial therapy could eliminate P. aeruginosa biofilms in a fish wound infection model, and enabled animal survival.

Conclusion: Our work provides a novel SILAC-based proteomics method that can simultaneously evaluate host and microbe proteomes, with future applications in higher host organisms and other microbial species. It also provides insights into the mechanisms dictating host-microbe interactions, offering novel strategies for anti-infective therapy.

Introduction: Lignin is a principal constituent of the secondary cell wall, which plays a role in both plant growth and defensing against stress, such as low temperature and pest infestation. Additionally, it also accumulates in fleshy fruits and negatively affects fruit quality. Red-fleshed loquat is temperature sensitive and exhibits cold-induced lignification. A number of technologies have been developed, for example, Low Temperature Conditioning (LTC) treatment, which has been applied in order to relieve the symptom of cold injury.

Objectives: The present study seeks to elucidate the regulatory mechanism underlying cold-induced lignification in loquat fruit.

Methods: The target genes were isolated through the analysis of transcriptome. The gene function was analyzed by transient transgenic method in tobacco leaves and loquat fruit, respectively, as well as stable overexpression in liverwort. The regulatory mechanism study was achieved by in vitro protein-protein interaction assays, dual-luciferase assay, and EMSA.

Results: In the present study, the Xylem NAC Domain transcription factor EjXND1 was identified as a repressor of loquat fruit lignification. It was demonstrated that EjXND1 could interact with the characterized lignin activator EjHB1, resulting in a diminution of the activation of EjHB1 on EjPRX12 promoter. Furthermore, two highly methylated regions were identified in the promoter of EjXDN1. One of these regions exhibited a negative correlation between methylation level and EjXND1 expression. Additionally, it was shown that hypermethylation of this region weaken the binding affinity of EjXND1 activators to its promoter.

Conclusion: The EjXND1 plays a role in modified Low Temperature Conditioning (mLTC) treatment that alleviates cold-induced lignification in red-fleshed loquat fruit by targeting the EjHB1-EjPRX12 module and EjXND1 is regulated by the dynamic of DNA methylation level in the promoter.

Introduction: Methyl jasmonate (MeJA) and MYB transcription factors (TFs) play important roles in pathogen resistance in several plants, but MYB TFs in conjunction with MeJA-induced defense against Pseudomonas tolaasii in edible mushrooms remain unknown.

Objectives: To investigate the role of a novel 3R-MYB transcription factor (AbMYB11) in MeJA-induced disease resistance of Agaricus bisporus and in the resistance of transgenic Arabidopsis to P. tolaasii.

Methods: Mushrooms were treated with MeJA alone or in combination with phenylpropanoid pathway inhibitors, and the effects of the treatments on the disease-related and physiological indicators of the mushrooms were determined to assess the role of MeJA in inducing resistance and the importance of the phenylpropanoid pathway involved. Subcellular localization, gene expression analysis, dual-luciferase reporter assay, electrophoretic mobility shift assay, and transgenic Arabidopsis experiments were performed to elucidate the molecular mechanism of AbMYB11 in regulating disease resistance.

Results: MeJA application greatly improved mushroom resistance to P. tolaasii infection, and suppression of the phenylpropanoid pathway significantly weakened this effect. MeJA treatment stimulated the accumulation of phenylpropanoid metabolites, which was accompanied by increased the activities of biosynthetic enzymes and the expression of phenylpropanoid pathway-related genes (AbPAL1, Ab4CL1, AbC4H1) and an AbPR-like gene, further confirming the critical role of the phenylpropanoid pathway in MeJA-induced responses to P. tolaasii. Importantly, AbMYB11, localized in the nucleus, was rapidly induced by MeJA treatment under P. tolaasii infection; it transcriptionally activated the phenylpropanoid pathway-related and AbPR-like genes, and AbMYB11 overexpression in Arabidopsis significantly increased the transcription of phenylpropanoid-related genes, the accumulation of total phenolics and flavonoids, and improved resistance to P. tolaasii.

Conclusion: This study clarified the pivotal role of AbMYB11 as a regulator in disease resistance by modulating the phenylpropanoid pathway, providing a novel idea for the breeding of highly disease-resistant edible mushrooms and plants.

Introduction: Developmental competence of oocytes matured in vitro is limited due to a lack of complete understanding of metabolism and metabolic gene expression during oocyte maturation and embryo development. Conventional metabolic analysis requires a large number of samples and is not efficiently applicable in oocytes and early embryos, thereby posing challenges in identifying key metabolites and regulating their in vitro culture system.

Objectives: To enhance the developmental competence of sheep oocytes, this study aimed to identify and supplement essential metabolites that were deficient in the culture systems.

Methods: The metabolic characteristics of oocytes and embryos were determined using ultrasensitive metabolomics analysis on trace samples and single-cell RNA-seq. By conducting integrated analyses of metabolites in cells (oocytes and embryos) and their developmental microenvironment (follicular fluid, oviductal fluid, and in vitro culture systems), we identified key missing metabolites in the in vitro culture systems. In order to assess the impact of these key missing metabolites on oocyte development competence, we performed in vitro culture experiments. Furthermore, omics analyses were employed to elucidate the underlying mechanisms.

Results: Our findings demonstrated that betaine, carnitine and creatine were the key missing metabolites in vitro culture systems and supplementation of betaine and L-carnitine significantly improved the blastocyst formation rate (67.48% and 48.61%). Through in vitro culture experiments and omics analyses, we have discovered that L-carnitine had the potential to promote fatty acid oxidation, reduce lipid content and lipid peroxidation level, and regulate spindle morphological grade through fatty acid degradation pathway. Additionally, betaine may participate in methylation modification and osmotic pressure regulation, thereby potentially improving oocyte maturation and early embryo development in sheep.

Conclusion: Together, these analyses identified key metabolites that promote ovine oocyte maturation and early embryo development, while also providing a new viewpoint to improve clinical applications such as oocyte maturation or embryo culture.

Introduction: Owing to the limited treatment options for hepatocellular carcinoma (HCC), interventions targeting pre-HCC stages have attracted increasing attention. In the pre-HCC stage, hepatic tumor-initiating cells (hTICs) proliferate abnormally and contribute to hepatocarcinogenesis. Numerous studies have investigated targeted senescence induction as an HCC intervention. However, it remains to be clarified whether senescence induction of hTICs could serve as a pre-HCC intervention.

Objectives: This study was designed to investigate whether senescence induction of hTICs in the precancerous stage inhibit HCC initiation.

Methods and results: HCC models developed from chronic liver injury (CLI) were established by using Fah^-/- mice and N-Ras + AKT mice. PD-0332991, a selective CDK4/6 inhibitor that blocks the G1/S transition in proliferating cells, was used to induce senescence during the pre-HCC stage. Upon administration of PD-0332991, we observed a significant reduction in HCC incidence following selective senescence induction in hTICs, and an alleviation liver injury in the CLI-HCC models. PD-0332991 also induced senescence in vitro in cultured hTICs isolated from CLI-HCC models. Moreover, RNA sequencing (RNA-seq) analysis delineated that the "Cyclin D-CDK4/6-INK4-Rb" pathway was activated in both mouse and human liver samples during the pre-HCC stage, while PD-0332991 exhibited substantial inhibition of this pathway, thereby inducing cellular senescence in hTICs. Regarding the immune microenvironment, we demonstrated that senescent hTICs secrete key senescence-associated secretory phenotypic (SASP) factors, CXCL10 and CCL2, to activate and recruit macrophages, and contribute to immune surveillance.

Conclusion: We found that hTICs can be targeted and induced into a senescent state during the pre-HCC stage. The SASP factors released by senescent hTICs further activate the immune response, facilitating the clearance of hTICs, and consequently suppressing HCC occurrence. We highlight the importance of pre-HCC interventions and propose that senescence-inducing drugs hold promise for preventing HCC initiation under CLI.

Introduction: Hepatocellular carcinoma (HCC) is a fatal cancer that is often diagnosed at the advanced stages which limits the available therapeutic options. The interaction of HGF with c-MET (a receptor tyrosine kinase) results in the activation of c-MET which subsequently triggers the PI3K/Akt/mTOR axis. Overexpression of c-MET in HCC tissues has been demonstrated to contribute to tumor progression and metastasis.

Objectives: We aimed to synthesize triazole-indirubin conjugates, examine their growth suppressor efficacy in cell-based assays, and investigate the antitumor as well as antimetastatic activity of lead cytotoxic agent in the orthotopic mice model.

Methods: A series of triazole-indirubin hybrids were synthesized and cytotoxicity, apoptogenic, and antimigratory effect of the lead compound (CRI9) was evaluated using MTT assay, cell cycle analysis, annexin-V/PI assay, TUNEL assay, and wound healing assay. The effect of CRI9 on the operation of the HGF/c-MET/PI3K/Akt/mTOR axis was examined using western blotting and transfection experiments. Acute toxicity, antitumor, and antimetastatic activity of CRI9 were examined in NCr nude mice. The expression of c-MET/PI3K/Akt/mTOR, CD31, and Ki-67 was examined using immunohistochemistry and western blotting.

Results: Among the new compounds, CRI9 consistently displayed potent cytotoxicity against HGF-induced HCC cells. CRI9 induced apoptosis as evidenced by increased sub G1 cells, annexin-V⁺/PI⁺ cells, TUNEL⁺ cells, and cleavage of procaspase-3 and PARP. CRI9 inhibited HGF-induced phosphorylation of c-MET^Y1234/1235 and subsequently suppressed the PI3K/Akt/mTOR axis. Also, depletion of c-MET or inhibition of c-MET by CRI9 resulted in suppression of the PI3K/Akt/mTOR axis. CRI9 showed no toxic effects in NCr nude mice and displayed a potent antitumor and antimetastatic effect in the orthotopic HCC mice model. CRI9 also reduced the levels of phospho-c-MET, CD31, and Ki-67 and suppressed the activation of the PI3K/Akt/mTOR axis in tumor tissues.

Conclusion: CRI9 has been identified as a new inhibitor of the c-MET/PI3K/Akt/mTOR axis in HCC preclinical models.

Introduction: Bacterial living states and the distribution of microbial colony signaling molecules are widely studied using mass spectrometry imaging (MSI). However, current approaches often treat 3D colonies as flat 2D disks, inadvertently omitting valuable details. The challenge of achieving 3D MSI in biofilms persists due to the unique properties of microbial samples.

Objectives: The study aimed to develop a new biofilm sample preparation method that can realize high-resolution 3D MSI of bacterial colonies to reveal the spatial organization of bacterial colonies.

Methods: This article introduces the moisture-assisted cryo-section (MACS) method, enabling embedding-free sectioning parallel to the growth plane. The MACS method secures intact sections by controlling ambient humidity and slice thickness, preventing molecular delocalization.

Results: Combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI)-MSI, the MACS method provides high-resolution insights into endogenic and exogenous molecule distributions in Pseudomonas aeruginosa (P. aeruginosa) biofilms, including isomeric pairs. Moreover, analyzed colonies are revived into 3D models, vividly depicting molecular distribution from inner to outer layers. Additionally, we investigated metabolite spatiotemporal dynamics in multiple colonies, observing changes over time and distinct patterns in single versus merged colonies. These findings shed light on the repel-merge process for multi-colony formation. Furthermore, our study monitored chemical responses inside biofilms after antibiotic treatment, showing increased antibiotic levels in the outer biofilm layer over time while maintaining low levels in the inner region. Moreover, the MACS method demonstrated its universality and applicability to other bacterial strains.

Conclusion: These results unveil complex cell activities within biofilm colonies, offering insights into microbe communities. The MACS method is universally applicable to loosely packed microorganism colonies, overcoming the limitations of previously reported MSI methods. It has great potential for studying bacterial-infected cancer tissues and artificial organs, making it a valuable tool in microbiological research.