Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.

Bacillus cereus NJ01 induces SA- and ABA-mediated immunity against bacterial pathogens through the EDS1-WRKY18 module.

Emerging evidence suggests a beneficial role of rhizobacteria in ameliorating plant disease resistance in an environment-friendly way. In this study, we characterize a rhizobacterium, Bacillus cereus NJ01, that enhances bacterial pathogen resistance in rice and Arabidopsis. Transcriptome analyses show that root inoculation of NJ01 induces the expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes in Arabidopsis leaves. Genetic evidence showed that EDS1, PAD4, and WRKY18 are required for B. cereus NJ01-induced bacterial resistance. An EDS1-PAD4 complex interacts with WRKY18 and enhances its DNA binding activity. WRKY18 directly binds to the W box in the promoter region of the SA biosynthesis gene ICS1 and ABA biosynthesis genes NCED3 and NCED5 and contributes to the NJ01-induced bacterial resistance. Taken together, our findings indicate a role of the EDS1/PAD4-WRKY18 complex in rhizobacteria-induced disease resistance.

Nanomaterials in 4D Printing: Expanding the Frontiers of Advanced Manufacturing.

As an innovative technology, four-dimentional (4D) printing is built upon the principles of three-dimentional (3D) printing with an additional dimension: time. While traditional 3D printing creates static objects, 4D printing generates "responsive 3D printed structures", enabling them to transform or self-assemble in response to external stimuli. Due to the dynamic nature, 4D printing has demonstrated tremendous potential in a range of industries, encompassing aerospace, healthcare, and intelligent devices. Nanotechnology has gained considerable attention owing to the exceptional properties and functions of nanomaterials. Incorporating nanomaterials into an intelligent matrix enhances the physiochemical properties of 4D printed constructs, introducing novel functions. This review provides a comprehensive overview of current applications of nanomaterials in 4D printing, exploring their synergistic potential to create dynamic and responsive structures. Nanomaterials play diverse roles as rheology modifiers, mechanical enhancers, function introducers, and more. The overarching goal of this review is to inspire researchers to delve into the vast potential of nanomaterial-enabled 4D printing, propelling advancements in this rapidly evolving field.

The development of a novel bio-based corrosion inhibitor: using biomass-derived 5-hydroxymethylfurfural (5-HMF) as a starting material.

An environmentally friendly corrosion inhibitor was prepared from the bio-based platform 5-hydroxymethylfurfural. This corrosion inhibitor was confirmed to be an efficient mixed-type corrosion inhibitor through a weight loss experiment and electrochemical experiment. Both thermodynamic and kinetic parameters were calculated and discussed, indicating that the adsorption of this bio-based inhibitor on a steel surface is a chemisorption process. Moreover, quantum chemical calculations were performed and further confirmed the formation of an effective productive film of this bio-based inhibitor on the metal surface. It is worth noting that the synthesis route of this bio-based corrosion inhibitor is green and environmentally friendly, and does not involve toxic chemical reagents.

WRR4B contributes to a broad-spectrum disease resistance against powdery mildew in Arabidopsis.

Oidium heveae HN1106, a powdery mildew (PM) that infects rubber trees, has been found to trigger disease resistance in Arabidopsis thaliana through ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-, PHYTOALEXIN DEFICIENT 4 (PAD4)- and salicylic acid (SA)-mediated signalling pathways. In this study, a typical TOLL-INTERLEUKIN 1 RECEPTOR, NUCLEOTIDE-BINDING, LEUCINE-RICH REPEAT (TIR-NB-LRR)-encoding gene, WHITE RUST RESISTANCE 4 (WRR4B), was identified to be required for the resistance against O. heveae in Arabidopsis. The expression of WRR4B was upregulated by O. heveae inoculation, and WRR4B positively regulated the expression of genes involved in SA biosynthesis, such as EDS1, PAD4, ICS1 (ISOCHORISMATE SYNTHASE 1), SARD1 (SYSTEMIC-ACQUIRED RESISTANCE DEFICIENT 1) and CBP60g (CALMODULIN-BINDING PROTEIN 60 G). Furthermore, WRR4B triggered self-amplification, suggesting that WRR4B mediated plant resistance through taking part in the SA-based positive feedback loop. In addition, WRR4B induced an EDS1-dependent hypersensitive response in Nicotiana benthamiana and contributed to disease resistance against three other PM species: Podosphaera xanthii, Erysiphe quercicola and Erysiphe neolycopersici, indicating that WRR4B is a broad-spectrum disease resistance gene against PMs.

Arabidopsis PHYTOALEXIN DEFICIENT 4 promotes the maturation and nuclear accumulation of immune-related cysteine protease RD19.

Arabidopsis PHYTOALEXIN DEFICIENT 4 (PAD4) has an essential role in pathogen resistance as a heterodimer with ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). Here we investigated an additional PAD4 role in which it associates with and promotes the maturation of the immune-related cysteine protease RESPONSIVE TO DEHYDRATION 19 (RD19). We found that RD19 and its paralog RD19c promoted EDS1- and PAD4-mediated effector-triggered immunity to an avirulent Pseudomonas syringae strain, DC3000, expressing the effector AvrRps4 and basal immunity against the fungal pathogen Golovinomyces cichoracearum. Overexpression of RD19, but not RD19 protease-inactive catalytic mutants, in Arabidopsis transgenic lines caused EDS1- and PAD4-dependent autoimmunity and enhanced pathogen resistance. In these lines, RD19 maturation to a pro-form required its catalytic residues, suggesting that RD19 undergoes auto-processing. In transient assays, PAD4 interacted preferentially with the RD19 pro-protease and promoted its nuclear accumulation in leaf cells. Our results lead us to propose a model for PAD4-stimulated defense potentiation. PAD4 promotes maturation and nuclear accumulation of processed RD19, and RD19 then stimulates EDS1-PAD4 dimer activity to confer pathogen resistance. This study highlights potentially important additional PAD4 functions that eventually converge on canonical EDS1-PAD4 dimer signaling in plant immunity.

4D Printed Nerve Conduit with In Situ Neurogenic Guidance for Nerve Regeneration.

Nerve repair poses a significant challenge in the field of tissue regeneration. As a bioengineered therapeutic method, nerve conduits have been developed to address damaged nerve repair. However, despite their remarkable potential, it is still challenging to encompass complex physiologically microenvironmental cues (both biophysical and biochemical factors) to synergistically regulate stem cell differentiation within the implanted nerve conduits, especially in a facile manner. In this study, a neurogenic nerve conduit with self-actuated ability has been developed by in situ immobilization of neurogenic factors onto printed architectures with aligned microgrooves. One objective was to facilitate self-entubulation, ultimately enhancing nerve repairs. Our results demonstrated that the integration of topographical and in situ biological cues could accurately mimic native microenvironments, leading to a significant improvement in neural alignment and enhanced neural differentiation within the conduit. This innovative approach offers a revolutionary method for fabricating multifunctional nerve conduits, capable of modulating neural regeneration efficiently. It has the potential to accelerate the functional recovery of injured neural tissues, providing a promising avenue for advancing nerve repair therapies.

The First High-Quality Chromosome-Level Genome of the Lutjanus erythropterus (Bloch, 1790) Using Single-Tube Long Fragment Reads and Hi-C Technologies.

Lutjanus erythropterus (Bloch, 1790), a Perciformes from the Lutjanidae family, is a commercially important species because of its taste and abundance. Despite the increase in genome resources in recent years, few genome assemblies are available within this fish family for comparative and functional studies. In this study, we determined the chromosomal genome of Crimson snapper using high-throughput Single-Tube Long Fragment Reads sequencing technology and Hi-C data. The final assembly size was 973.04 Mb with contig and scaffold N50 values of 1.51 and 40.65 Mb, respectively. We successfully scaffolded 95.84% of the genome sequence onto 24 chromosomes ranging in length from 19.37 to 49.48 Mb. A total of 22,663 genes and 13,877 gene families were identified in the genome, with 29 gene families being L. erythropterus-specific. A phylogenetic analysis using single-copy gene families showed that L. erythropterus and Larimichthys crocea had the closest genetic relationship with a divergence time of ∼47.7 Ma. This new genomic resource will facilitate comparative genomic studies as well as genetic breeding programs for L. erythropterus.

3D printing of thick myocardial tissue constructs with anisotropic myofibers and perfusable vascular channels.

Engineering of myocardial tissues has become a promising therapeutic strategy for treating myocardial infarction (MI). However, a significant challenge remains in generating clinically relevant myocardial tissues that possess native microstructural characteristics and fulfill the requirements for implantation within the human body. In this study, a thick 3D myocardial construct with anisotropic myofibers and perfusable branched vascular channels is created with clinically relevant dimensions using a customized beam-scanning stereolithography printing technique. To obtain tissue-specific matrix niches, a decellularized extracellular matrix microfiber-reinforced gelatin-based bioink is developed. The bioink plays a crucial role in facilitating the precise manufacturing of a hierarchical microstructure, enabling us to better replicate the physiological characteristics of the native myocardial tissue matrix in terms of structure, biomechanics, and bioactivity. Through the integration of the tailored bioink with our printing method, we demonstrate a biomimetic architecture, appropriate biomechanical properties, vascularization, and improved functionality of induced pluripotent stem cell-derived cardiomyocytes in the thick tissue construct in vitro. This work not only offers a novel and effective means to generate biomimetic heart tissue in vitro for the treatment of MI, but also introduces a potential methodology for creating clinically relevant tissue products to aid in other complex tissue/organ regeneration and disease model applications.

4D Thermo-Responsive Smart hiPSC-CM Cardiac Construct for Myocardial Cell Therapy.

Purpose: 4D fabrication techniques have been utilized for advanced biomedical therapeutics due to their ability to create dynamic constructs that can transform into desired shapes on demand. The internal structure of the human cardiovascular system is complex, where the contracting heart has a highly curved surface that changes shape with the heart's dynamic beating motion. Hence, 4D architectures that adjust their shapes as required are a good candidate to readily deliver cardiac cells into the damaged heart and/or to serve as self-morphing tissue scaffolds/patches for healing cardiac diseases. In this proof-of-concept in vitro study, a two-in-one 4D smart cardiac construct that integrates the functions of minimally invasive cell vehicles and in situ tissue patches was developed for repairing damaged myocardial tissue. Methods: For this purpose, a series of thermo-responsive 4D structures with different shapes and sizes were fabricated via the combination of fused deposition modeling (FDM)-printing and stamping molding. The thermo-responsive 4D constructs were firstly optimized to exhibit their shape transformation behavior at the designated temperature for convenient control. After which, the mechanical properties, shape recovery rate, and shape recovery speed of the 4D constructs at different temperatures were thoroughly evaluated. Also, the proliferation and functional prototype of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on the 4D constructs were quantified and evaluated using F-actin staining and immunostaining. Results: Our results showed that the 4D constructs possessed the desirable capability of shape-changing from spherical carriers to unfolded patches at human body temperature and exhibited excellent biocompatibility. Moreover, myocardial maturation in vitro with a uniform and printing pattern-specific cell distribution was observed on the surface of the unfolded 4D constructs. Conclusion: We successfully developed a 4D smart cardiac construct that integrates the functions of minimally invasive cell vehicles and in situ tissue patches for repairing damaged myocardial tissue.

ARBUSCULAR MYCORRHIZA-INDUCED KINASES AMK8 and AMK24 associate with the receptor-like kinase KINASE3 to regulate arbuscular mycorrhizal symbiosis in Lotus japonicus.

Arbuscular mycorrhizal (AM) symbiosis is a widespread, ancient mutualistic association between plants and fungi, and facilitates nutrient uptake into plants. Cell surface receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) play pivotal roles in transmembrane signaling, while few RLCKs are known to function in AM symbiosis. Here, we show that 27 out of 40 AM-induced kinases (AMKs) are transcriptionally upregulated by key AM transcription factors in Lotus japonicus. Nine AMKs are only conserved in AM-host lineages, among which the SPARK-RLK-encoding gene KINASE3 (KIN3) and the RLCK paralogues AMK8 and AMK24 are required for AM symbiosis. KIN3 expression is directly regulated by the AP2 transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), which regulates the reciprocal exchange of nutrients in AM symbiosis, via the AW-box motif in the KIN3 promoter. Loss of function mutations in KIN3, AMK8, or AMK24 result in reduced mycorrhizal colonization in L. japonicus. AMK8 and AMK24 physically interact with KIN3. KIN3 and AMK24 are active kinases and AMK24 directly phosphorylates KIN3 in vitro. Moreover, CRISPR-Cas9-mediated mutagenesis of OsRLCK171, the sole homolog of AMK8 and AMK24 in rice (Oryza sativa), leads to diminished mycorrhization with stunted arbuscules. Overall, our results reveal a crucial role of the CBX1-driven RLK/RLCK complex in the evolutionarily conserved signaling pathway enabling arbuscule formation.

Difluorocarbene-Triggered Acyl Rearrangement Reaction: A Strategy for the Direct Introduction of the gem-Difluoromethylene Group.

A novel metal- and catalyst-free dearomative reaction of 2-oxypyridines to construct gem-difluoromethylenated N-substituted 2-pyridones has been developed. The reaction involves an attractive acyl rearrangement from O to CF2 of difluorocarbene-derived pyridinium ylides, which provides a new strategy for the direct introduction of the gem-difluoromethylene group with high efficiency and selectivity as well as broad substrate scope. Gram-scale synthesis and synthetic transformations have also been demonstrated.

3D printed biomimetic flexible blood vessels with iPS cell-laden hierarchical multilayers.

Successful recovery from vascular diseases has typically relied on the surgical repair of damaged blood vessels (BVs), with the majority of current approaches involving the implantation of autologous BVs, which is plagued by donor site tissue damage. Researchers have attempted to develop artificial vessels as an alternative solution to traditional approaches to BV repair. However, the manufacturing of small-diameter (< 6 mm) BVs is still considered one of the biggest challenges due to its difficulty in the precise fabrication and the replication of biomimetic architectures. In this study, we successfully developed 3D printed flexible small-diameter BVs that consist of smooth muscle cells and a vascularized endothelium. In the developed artificial BV, a rubber-like elastomer was printed as the outermost layer of the vessel, which demonstrated enhanced mechanical properties, while and human induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iSMCs) and endothelial cells (iECs) embedded fibrinogen solutions were coaxially extruded with thrombin solution to form cell-laden fibrin gel inner layers. Our results showed that the 3D BVs possessed proper mechanical properties, and the cells in the fibrin layers substantially proliferated over time to form a stable BV construct. Our study demonstrated that the 3D printed flexible small-diameter BV using iPSCs could be a promising platform for the treatment of vascular diseases.

An in vitro analysis of the effect of geometry-induced flows on endothelial cell behavior in 3D printed small-diameter blood vessels.

Clinical recovery from vascular diseases has increasingly become reliant upon the successful fabrication of artificial blood vessels (BVs) or vascular prostheses due to the shortage of autologous vessels and the high incidence of vessel graft diseases. Even though many attempts at the clinical implementation of large artificial BVs have been reported to be successful, the development of small-diameter BVs remains one of the significant challenges due to the limitation of micro-manufacturing capacity in complexity and reproducibility, as well as the development of thrombosis. The present study aims to develop 3D printed small-diameter artificial BVs that recapitulate the longitudinal geometric elements in the native BVs using biocompatible polylactic acid (PLA). As their intrinsic physical properties are crystallinity dependent, we used two PLA filaments with different crystallinity to investigate the suitability of their physical properties in the micro-manufacturing of BVs. To explore the mechanism of venous thrombosis, our study provided a preliminarily comparative analysis of the effect of geometry-induced flows on the behavior of human endothelial cells (ECs). Our results showed that the adhered healthy ECs in the 3D printed BV exhibited regulated patterns, such as elongated and aligned parallel to the flow direction, as well as geometry-induced EC response mechanisms that are associated with hemodynamic shear stresses. Furthermore, the computational fluid dynamics simulation results provided insightful information to predict velocity profile and wall shear stress distribution in the geometries of BVs in accordance with their spatiotemporally-dependent cell behaviors. Our study demonstrated that 3D printed small-diameter BVs could serve as suitable candidates for fundamental BV studies and hold great potential for clinical applications.

Circular RNA hsa_circ_0083756 promotes intervertebral disc degeneration by sponging miR-558 and regulating TREM1 expression.

OBJECTIVES: Intervertebral disc degeneration (IVDD) is a leading cause of low back pain. Circular RNAs (circRNAs) have been demonstrated to exert vital functions in IVDD. However, the role and mechanism of hsa_circ_0083756 in the development of IVDD remain unclear. MATERIALS AND METHODS: RT-qPCR was performed to detect expressions of hsa_circ_0083756, miR-558 and TREM1 in nucleus pulposus (NP) tissues and cells. CCK8 assay, flow cytometry, TUNEL assay, RT-qPCR and WB were used to clarify the roles of hsa_circ_0083756 in NP cells proliferation and extracellular matrix (ECM) formation. Bioinformatics analyses, dual-luciferase reporter gene experiment, RNA immunoprecipitation (RIP) assay and FISH assay were performed to predict and verify the targeting relationship between hsa_circ_0083756 and miR-558, as well as that between miR-558 and TREM1. Ultimately, the effect of hsa_circ_0083756 on IVDD was tested through anterior disc-puncture IVDD animal model in rats. RESULTS: hsa_circ_0083756 was upregulated in degenerative NP tissues and cells. In vitro loss-of-function and gain-of-function studies suggested that hsa_circ_0083756 knockdown promoted, whereas hsa_circ_0083756 overexpression inhibited NP cells proliferation and ECM formation. Mechanistically, hsa_circ_0083756 acted as a sponge of miR-558 and subsequently promoted the expression of TREM1. Furthermore, in vivo study indicated that silencing of hsa_circ_0083756 could alleviate IVDD in rats. CONCLUSIONS: hsa_circ_0083756 promoted IVDD via targeting the miR-558/TREM1 axis, and hsa_circ_0083756 may serve as a potential therapeutic target for the treatment of IVDD.

Influence of incentive mechanism and fit degree on user's environmental behavior-Taking Alipay "Ant Forest" in China as an example.

How to use game elements to motivate users and influence their behavior has become a new research trend, which is vital for enhancing the willingness of potential platform users to participate in environmental protection. This paper aims to analyze the influence of incentive mechanism and fit degree on user's environmental behavior based on the stimulus-organism-response theory and self-determination theory. The questionnaire data of 500 users was collected and the impact of incentives on user's environmental behavior was analyzed by structural equation modeling. The results show that economic, value, and social incentives have a significant impact on user's environmental behavior. Besides, the value and social incentives of "Ant Forest" game platform positively influence user fit (conscious participation, enthusiasm, and platform interaction), but the impact of economic incentive on platform interaction is not statistically significant. From the perspective of user fit, "Ant Forest" game platform can positively promote users to adopt environmental behavior, because it explores users' needs from their perspective to give full play to the role of game incentives on users' environmental behavior. Additionally, this research provides the practical implications for managers exploring the effects of co-creation processes in developing countries and regions.

A sugarcane smut fungus effector simulates the host endogenous elicitor peptide to suppress plant immunity.

The smut fungus Sporisorium scitamineum causes the most prevalent disease on sugarcane. The mechanism of its pathogenesis, especially the functions and host targets of its effector proteins, are unknown. In order to identify putative effectors involving in S. scitamineum infection, a weighted gene co-expression network analysis was conducted based on the transcriptome profiles of both smut fungus and sugarcane using a customized microarray. A smut effector gene, termed SsPele1, showed strong co-expression with sugarcane PLANT ELICITOR PEPTIDE RECEPTOR1 (ScPEPR1), which encodes a receptor like kinase for perception of plant elicitor peptide1 (ScPep1). The relationship between SsPele1 and ScPEPR1, and the biological function of SsPele1 were characterized in this study. The SsPele1 C-terminus contains a plant elicitor peptide-like motif, by which SsPele1 interacts strongly with ScPEPR1. Strikingly, the perception of ScPep1 on ScPEPR1 is competed by SsPele1 association, leading to the suppression of ScPEPR1-mediated immune responses. Moreover, the Ustilago maydis effector UmPele1, an ortholog of SsPele1, promotes fungal virulence using the same strategy. This study reveals a novel strategy by which a fungal effector can mimic the plant elicitor peptide to complete its perception and attenuate receptor-activated immunity.

A new biochemistry connecting pathogen detection to induced defense in plants.

Plant cell surface and intracellular immune receptors recognizing pathogen attack utilize the same defense machineries to mobilize resistance. New genetic, protein structural and biochemical information on receptor activation and signaling is transforming understanding of how their shared defense network operates. We discuss the biochemical activities of two classes of intracellular nucleotide-binding/leucine-rich repeat (NLR) receptor - one forming a Ca2+ channel, the other an NADase enzyme - which define engagement of enhanced disease susceptibility 1 (EDS1)-family heterodimers and cofunctioning helper NLRs (RNLs) to connect receptor systems and amplify defenses. Toll-interleukin-1 receptor (TIR) domain NLR receptors and TIR-domain proteins, with a capacity to produce NAD+-derived small molecules, require EDS1 dimers and RNLs for defense induction. The TIR-driven EDS1/RNL modules emerge as central elements in Ca2+ -based immunity signaling initiated by receptors outside and inside host cells.

Emerging 4D Printing Strategies for Next-Generation Tissue Regeneration and Medical Devices.

The rapid development of 3D printing has led to considerable progress in the field of biomedical engineering. Notably, 4D printing provides a potential strategy to achieve a time-dependent physical change within tissue scaffolds or replicate the dynamic biological behaviors of native tissues for smart tissue regeneration and the fabrication of medical devices. The fabricated stimulus-responsive structures can offer dynamic, reprogrammable deformation or actuation to mimic complex physical, biochemical, and mechanical processes of native tissues. Although there is notable progress made in the development of the 4D printing approach for various biomedical applications, its more broad-scale adoption for clinical use and tissue engineering purposes is complicated by a notable limitation of printable smart materials and the simplistic nature of achievable responses possible with current sources of stimulation. In this review, the recent progress made in the field of 4D printing by discussing the various printing mechanisms that are achieved with great emphasis on smart ink mechanisms of 4D actuation, construct structural design, and printing technologies, is highlighted. Recent 4D printing studies which focus on the applications of tissue/organ regeneration and medical devices are then summarized. Finally, the current challenges and future perspectives of 4D printing are also discussed.