Journal of advanced research最新文献

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.

Introduction: Type I interferon (IFN-I, IFN-α/β), precisely controlled by multiple regulators, including suppressor of cytokine signaling 1 (SOCS1), is critical for host defense against pathogens. However, the impact of IFN-α/β on malaria parasite infections, beneficial or detrimental, remains controversial.

Objectives: The contradictory results are suspected to arise from differences in parasite species and host genetic backgrounds. To date, no prior study has employed a comparative approach utilizing two parasite models to investigate the underlying mechanisms of IFN-I response. Moreover, whether and how SOCS1 involves in the distinct IFN-α/β dynamics is still unclear.

Methods: Here we perform single-cell RNA sequencing analyses (scRNA-seq) to dissect the dynamics of IFN-α/β responses against P. yoelii 17XL (17XL) and P. berghei ANKA (PbANKA) infections; conduct flow cytometry analysis and functional depletion to identify key cellular players induced by IFN-I; and establish mathematical models to explore the mechanisms underlying the differential IFN-I dynamics regulated by SOCS1.

Results: 17XL stimulates an early protective but insufficient toll-like receptor 7 (TLR7)-interferon regulatory factor 7 (IRF7)-dependent IFN-α/β response, resulting in CD11a^hiCD49d^hiCD4⁺ T cell activation to enhance anti-malarial immunity. On the contrary, a late IFN-α/β induction through toll-like receptor 9 (TLR9)-IRF7/ stimulator of interferon genes (STING)- interferon regulatory factor 3 (IRF3) dependent pathways expands programmed cell death protein 1 (PD-1)⁺CD8⁺ T cells and impairs host immunity during PbANKA infection. Furthermore, functional assay and mathematical modeling show that SOCS1 significantly suppresses IFN-α/β production via negative feedback and incoherent feed-forward loops (I1-FFL). Additionally, differential activation patterns of various transcriptional factors (TFs) synergistically regulate the distinct IFN-I responses.

Conclusion: This study reveals the dual functions of IFN-I in anti-malarial immunity: Early IFN-α/β enhances immune responses against Plasmodium infection by promoting CD11a^hiCD49d^hiCD4⁺ T cell, while late IFN-α/β suppresses these response by expanding PD-1⁺CD8⁺ T cells. Moreover, both the SOCS1-related network motifs and TFs activation patterns contribute to determine distinct dynamics of IFN-I responses. Hence, our findings suggest therapies targeting SOCS1- or TFs-regulated IFN-I dynamics could be an efficacious approach for preventing malaria and enhancing vaccine efficacy.

Introduction: Metabolic syndrome is a serious public health concern across the globe. However, the typical metabolites and mechanisms underlying the decreased fertility related to metabolic syndrome is still elusive.

Objectives: The aim of the present study was to explore the typical metabolites and mechanisms underlying the decreased fertility related with metabolic syndrome.

Methods: Utilizing metabolomics, a comparative analysis was conducted on fatty acid compositions in various tissues of sows with high and low reproductive performance. Additionally, serum fatty acid compositions in a metabolic syndrome model (obese mice) induced by a high-fat diet (HFD) were investigated to elucidate the lipid metabolites associated with metabolic syndrome. Furthermore, the impact of nervonic acid (NA) on ovarian function was examined using rodent animal models (rats and mice). Through biological techniques such as transcriptomics, CUT&Tag, and analysis of post-translational protein modifications, the molecular mechanisms underlying NA mediated ovarian inflammation were further elucidated based on models utilizing ovarian granulosa cells from pigs, humans, and mice. Finally, validation was performed on ovaries from patients diagnosed with polycystic ovary syndrome.

Results: In vitro, targeted serum lipidomic analysis revealed that sows with low embryo survival rates exhibited abnormal lipid metabolism characterized by abnormal accumulation of NA in the liver, ovary, and adipose tissue. Additionally, elevated NA levels trigger ovarian inflammation to cause ovarian dysfunction in both sows and rats. Mechanistically, NA induce mitochondrial oxidative stress through inhibiting respiratory chain proteins CYTB and NDFUB8 to activate NLRP3 inflammasome, which triggers procaspase-1 into active caspase-1, and convert the cytokine precursors pro-IL-1β into biologically active IL-1β in ovarian granulosa cells. Notably, we evidenced that NA promotes IL-1β activities by increasing H3K9ac modification level of IL-1β promoter regions and regulating the expression of the transcription factor AP-1. Finally, we found that the decreased expression of CerS2 in ovaries and the increased level of chemokine CXCL14 may be the cause of abnormal NA accumulation. Surprisingly, individuals with polycystic ovary syndrome, obesity, non-alcoholic fatty liver or gestational diabetes mellitus exhibit a high level of serum NA.

Conclusion: Collectively, our current study suggests that NA is a typical metabolite of metabolic syndrome, which strongly influences the ovarian function and embryo survival and also provides that interfering with mitochondrial ROS production is a potential strong strategy for target solving abnormal NA accumulation.

Introduction: Bentazon (BNTZ) is a selective contact herbicide widely used to control field weeds for crop production. Excessive use of BNTZ leads to its accumulation in soils and crops, becoming an environmental contaminant. Therefore, investigation of the mechanisms for BNTZ detoxification and degradation in crops is fundamentally important to reduce crop contamination and ensure food safety.

Objectives: This study aims to elucidate the mechanism of detoxification and degradation pathways of the BNTZ complex in rice by creating transgenic lines expressing a rice ATP-binding cassette (OsABC) transporter gene through genetic engineering techniques combined with chemical analytical techniques and metabolomics approaches.

Methods: We established the rice transgenic lines overexpressing (OE) a rice OsABC transporter and its knockout lines by CRISPR-Cas9 to characterize the gene function and measured the accumulation of BNTZ residues in rice. The metabolites of BNTZ were characterized by LC/Q-TOF-HRMS/MS (Liquid chromatography/time of flight-high resolution mass spectrometry).

Results: Overexpression of OsABC significantly conferred rice resistance to BNTZ toxicity by increasing plant elongation, dry weight, and chlorophyll content, and significantly reducing cell membrane damage and BNTZ accumulation in rice tissues. Six different metabolites and ten conjugates were well defined in chemical structures. The reduced BNTZ levels and degradation products in the grains of the OE lines supported the robust activity of the OsABC gene function. Using UPLC-Q-TOF/MS, we further identified accumulated basic metabolites of various carbohydrates, amino acids, hormones, and flavonoids, and found that these metabolites involved in BNTZ degradation were increased more in OE lines than in wild-type (WT) rice.

Conclusions: Our work demonstrates that the OsABC transporter plays a critical role in regulating the mobility and degradative metabolism of BNTZ in rice, thus revealing a regulatory mechanism underlying rice resistance to BNTZ toxicity and adaptation to the environmental stress.

Introduction: Wound infections and formation of biofilms caused by multidrug-resistant bacteria have constituted a series of wound deteriorated and life-threatening problems. The in situ resisting bacterial adhesion, killing multidrug-resistance bacteria, and releasing dead bacteria is strongly required to supply a gap of existing sterilization strategies.

Objectives: This study aims to present a facile approach to construct a bacteria-responsive hydrogel with switchable antimicrobial-antifouling properties through a "resisting-killing-releasing" method.

Methods: The smart bacteria-responsive hydrogel was constructed by two-step immersion strategy: a simple immersion-coating process to construct Polydopamine (pDA) coatings on the surface of a gelatin-chitosan composite hydrogel and followed by grafting of bactericidal quaternary ammonium chitosan (QCS) as well as pH-responsive PMAA to this pDA coating. The in vitro antimicrobial activity, biocompatibility and the in vivo wound healing effects in a mouse MRSA-infected full-thickness defect model of the hydrogel were further evaluated.

Results: Assisted by polydopamine coating, the pH-responsive PMAA and bactericidal QCS are successfully grafted onto a gelatin-chitosan composite hydrogel surface and hydrogels maintain the adequate mechanical properties. At physiological conditions, the PMAA hydration layer endows the hydrogel with resistance to initial bacterial attachment. Once bacteria colonize and acidize local environment, the swelling PMAA chains tend to collapse then expose the bactericidal QCS, realizing the on-demand kill bacteria. Moreover, the dead bacteria can be released and the hydrogel will resume the resistance due to hydrophilicity of PMAA at increased pH, endowing the surface renewable ability. In vitro and in vivo studies demonstrate the favorable biocompatibility and wound healing capacity of hydrogels that can inhibit infection and further facilitate granulation tissue, angiogenesis, and collagen synthesis.

Conclusion: This strategy provides a novel methodology for the development and design of smart wound dressing to combat multidrug-resistant bacteria infections.

Introduction: Patients with mantle cell lymphoma (MCL) frequently develop resistance to ibrutinib. Lymphoma-associated macrophages (LAMs) may play a causal role in this resistance but remain underexplored in current literature.

Objectives: To elucidate the role of LAMs in mediating ibrutinib resistance in MCL.

Methods: We investigated macrophage polarization through multiparameter flow cytometry (MPFC) using antibodies against CD206 and CD86 in blood and tissue samples from patients with MCL, both resistant and sensitive to ibrutinib. Subsequently, we developed an in vitro co-culture model utilizing MCL cell lines to identify cytokines associated with ibrutinib resistance and macrophage M2 polarization. The mechanisms underlying resistance were examined using MPFC, RNA sequencing, and Western blot analysis. Additionally, we assessed whether SB225002, a CXCR2 inhibitor, could reverse ibrutinib resistance through CCK-8 and caspase-3 assays, as well as in a mouse xenograft model involving an ibrutinib-resistant MCL cell line.

Results: In patients exhibiting ibrutinib resistance, the ratio of M2 to M1 LAMs was significantly higher compared to sensitive patients. In co-cultures of LAMs and MCL cells, the percentage of M2 macrophages, the IC50 value for ibrutinib, and the concentrations of IL-8 and CXCL5 were significantly elevated. Mechanistically, CXCL5 secreted by LAMs interacted with the CXCR2 on MCL cells, leading to the activation of the Akt, p38, and STAT3 signaling pathways in the presence of ibrutinib; this activity was diminished upon blockade of the CXCL5/CXCR2 axis. The combination of SB225002 and ibrutinib significantly enhanced MCL cell apoptosis, suppressed lymphoma growth in the xenograft model, and reprogrammed macrophage phenotype compared to treatment with ibrutinib alone.

Conclusion: Our data indicate that M2-polarized LAMs are associated with ibrutinib resistance in a model of MCL, and that a CXCR2 inhibitor can reverse this resistance. These findings suggest a potential new therapeutic strategy.

Background: Soybean is a worldwide-cultivated crop due to its applications in the food, feed, and biodiesel industries. Genome editing in soybean began with ZFN and TALEN technologies; however, CRISPR/Cas has emerged and shortly became the preferable approach for soybean genome manipulation since it is more precise, easy to handle, and cost-effective. Recent reports have focused on the conventional Cas9 nuclease, Cas9 nickase (nCas9) derived base editors, and Cas12a (formally Cpf1) as the most commonly used genome editors in soybean. Nonetheless, several challenges in the complex plant genetic engineering pipeline need to be overcome to effectively edit the genome of an elite soybean cultivar. These challenges include (1) optimizing CRISPR cassette design (i.e., gRNA and Cas promoters, gRNA design and testing, number of gRNAs, and binary vector), (2) improving transformation frequency, (3) increasing the editing efficiency ratio of targeted plant cells, and (4) improving soybean crop production.

Aim of review: This review provides an overview of soybean genome editing using CRISPR/Cas technology, discusses current challenges, and highlights theoretical (insights) and practical suggestions to overcome the existing bottlenecks.

Key scientific concepts of review: The CRISPR/Cas system was discovered as part of the bacterial innate immune system. It has been used as a biotechnological tool for genome editing and efficiently applied in soybean to unveil gene function, improve agronomic traits such as yield and nutritional grain quality, and enhance biotic and abiotic stress tolerance. To date, the efficiency of gRNAs has been validated using protoplasts and hairy root assays, while stable plant transformation relies on Agrobacterium-mediated and particle bombardment methods. Nevertheless, most steps of the CRISPR/Cas workflow require optimizations to achieve a more effective genome editing in soybean plants.