Journal of advanced research最新文献

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.

Introduction: Homocysteine (Hcy) is well recognized to be an independent risk factor for atherosclerosis. Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis, while the underlying mechanisms of its involvement in Hcy induced-atherosclerosis remain largely unknown.

Objectives: The primary aim of this study is to assess the role of lncARF (autophagy-related factor induced by Hcy) in Hcy induced-atherosclerosis and related mechanism.

Methods: RNA sequencing of foam cells treated with Hcy revealed a novel specific long noncoding RNA called lncARF. Locked nucleic acid gapmeRs-mediated lncARF knockdown was used to explore the role of lncARF both in vivo and in vitro. Mass spectrometry, RNA pull-down and RNA immunoprecipitation (RIP) assays were employed to uncover a mechanistic role of lncARF. Mass array assay and chromatin immunoprecipitation (ChIP) were used to detect the transcriptional activation of lncARF mediated by transcription factor. Clinically, receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic value of lncARF in atherosclerotic patients with hyperhomocysteinemia (HHcy).

Results: We observed that the expression of lncARF was substantially upregulated in atherosclerotic plaques, and knockdown of lncARF decreased the formation of atherosclerotic lesions by promoting autophagy in foam cells. Mechanistically, lncARF physically binds to RRAGD and inhibits its ubiquitination, further activating the PI3K/Akt and MAPK signaling pathways. Moreover, in vitro experiments showed that transcription factor FosB inhibited the binding of DNMT1 at the lncARF promoter, leading to transcriptional activation through DNA hypomethylation. Clinically, lncARF expression was positively correlated with serum Hcy levels, and it could distinguish atherosclerotic patients with HHcy with a high area under the ROC curve, sensitivity and specificity.

Conclusions: Our study highlights the mechanisms of lncARF in protecting against the development of atherosclerosis involving the epigenetic modifications and RRAGD/PI3K/Akt and RRAGD/MAPK signaling pathways, which may provide novel diagnostic biomarkers to improve atherosclerosis treatment.

Introduction: Simultaneous detection of proteins and mRNA within a single extracellular vesicle (EV) enables comprehensive analysis of specific EVs subpopulations, significantly advancing cancer diagnostics. However, developing a sensitive and user-friendly approach for simultaneously detecting multidimensional biomarkers in single EV is still challenging.

Objectives: To facilitate the analysis of multidimensional biomarkers in EVs and boost its clinical application, we present a versatile droplet digital system facilitating the concurrent detection of membrane proteins and mRNA at the single EV level with high sensitivity and specificity.

Methods: The antibody-DNA conjugates were firstly prepared for EVs protein biomarkers recognition and signal transformation. Coupling with the assembled triplex droplet digital PCR system, a versatile droplet digital analysis assay for simultaneous detection of membrane protein and mRNA at a single EV level was developed.

Results: Our new droplet digital system displayed high sensitivity and specificity. Additionally, its clinical application was validated in a breast cancer cohort. As expected, this assay has demonstrated superior performance in distinguishing breast cancer from healthy individuals and benign controls through combined detection of EVs protein and mRNA markers compared to any single kind marker detections, especially for patients with breast cancer at early stage (AUC=0.9229).

Conclusion: Consequently, this study proposes a promising strategy for accurately identifying and analyzing specific EV subgroups through the co-detection of proteins and mRNA at the single EV level, holding significant potential for future clinical applications.

Introduction: Extensive studies have established the correlation between long-term PM_2.5 exposure and lung cancer, yet the mechanisms underlying this association remain poorly understood. PIWI-interacting RNAs (piRNAs), a novel category of small non-coding RNAs, serve important roles in various diseases. However, their biological function and mechanism in PM_2.5-induced lung cancer have not been thoroughly investigated.

Objectives: We aimed to explore the oncogenic role of piRNA in lung cancer induced by PM_2.5 exposure, as well as the underlying mechanisms.

Methods: We conducted a PM_2.5-induced human lung epithelial cell malignant transformation model. Human samples were used to further verify the finding. In vitro proliferation, migration, and invasion assays were performed to study the function of piRNA. RNA-sequencing was used to elucidate the the mechanisms of how piRNA mediates cell functions. PiRNA pull-down and computational docking analysis were conducted to identify proteins that binding to piRNA. In vivo experiments were used to explore whether inhibition of PMLCPIR could have a therapeutic effect on lung cancer.

Results: We identified a new up-regulated piRNA, termed PM_2.5-induced lung cancer up-regulation piRNA (PMLCPIR), which promotes the proliferation of PM_2.5-transformed cells and lung cancer cells. RNA sequencing revealed ITGB1 as a downstream target of PMLCPIR. Importantly, PMLCPIR binds to nucleolin (NCL) and increases the expression of its target gene, ITGB1, thereby activating PI3K/AKT signaling. The inhibition of PMLCPIR could promote apoptosis in lung cancer cells and enhance their chemosensitivity to anti-tumor drugs.

Conclusion: We systematically identified the alterations of piRNA expression profiles in the PM_2.5-induced malignant transformation model. Then, PMLCPIR was recognized as a novel oncogenic piRNA in PM_2.5-induced lung cancer. Mechanically, PMLCPIR binds to NCL, enhancing ITGB1 expression and activating the ontogenetic PI3K/AKT signaling, potentially contributing to lung cancer progression. This study provides novel insights into the revelation of a new epigenetic regulator in PM_2.5-induced lung cancer.