Initial IL-10 production dominates the therapy of mesenchymal stem cell scaffold in spinal cord injury.

Rationale: Spinal cord injury (SCI) is an acute damage to the central nervous system that results in severe morbidity and permanent disability. Locally implanted scaffold systems with immobilized mesenchymal stem cells (MSCs) have been widely proven to promote locomotor function recovery in SCI rats; however, the underlying mechanism remains elusive. Methods and Results: In this study, we constructed a hyaluronic acid scaffold system (HA-MSC) to accelerate the adhesive growth of human MSCs and prolong their survival time in SCI rat lesions. MSCs regulate local immune responses by upregulating the expression of anti-inflammatory cytokines. Interestingly, the dramatically increased, but transient expression of interleukin 10 (IL-10) is found to be secreted by MSCs in the first week. Blocking the function of the initially produced IL-10 by the antibody completely abolished the neurological and behavioral recovery of SCI rats, indicating a core role of IL-10 in SCI therapy with HA-MSC implantation. Transcriptome analyses indicated that IL-10 selectively promotes the migration and cytokine secretion-associated programs of MSCs, which in turn helps MSCs exert their anti-inflammatory therapeutic effects. Conclusion: Our findings highlight a novel role of IL-10 in regulating MSC migration and cytokine secretion-associated programs, and determine the vital role of IL-10 in the domination of MSC treatment for spinal cord repair.

A high-efficient protoplast transient system for screening gene editing elements in Salvia miltiorrhiza.

KEY MESSAGE: A high-efficiency protoplast transient system was devised to screen genome editing elements in Salvia miltiorrhiza. Medicinal plants with high-value pharmaceutical ingredients have attracted research attention due to their beneficial effects on human health. Cell wall-free protoplasts of plants can be used to evaluate the efficiency of genome editing mutagenesis. The capabilities of gene editing in medicinal plants remain to be fully explored owing to their complex genetic background and shortfall of suitable transformation. Here, we took the Salvia miltiorrhiza as a representative example for developing a method to screen favorable gene editing elements with high editing efficiency in medical plants by a PEG-mediated protoplast transformation. Results indicated that using the endogenous SmU6.1 of S. miltiorrhiza to drive sgRNA and the plant codon-optimized Cas9 driven by the promoter SlEF1α can enhance the efficiency of editing. In summary, we uncover an efficacious transient method for screening editing elements and shed new light on increasing gene editing efficiency in medicinal plants.

Application of serum Raman spectroscopy combined with classification model for rapid breast cancer screening.

Introduction: This study aimed to evaluate the feasibility of using general Raman spectroscopy as a method to screen for breast cancer. The objective was to develop a machine learning model that utilizes Raman spectroscopy to detect serum samples from breast cancer patients, benign cases, and healthy subjects, with puncture biopsy as the gold standard for comparison. The goal was to explore the value of Raman spectroscopy in the differential diagnosis of breast cancer, benign lesions, and healthy individuals. Methods: In this study, blood serum samples were collected from a total of 333 participants. Among them, there were 129 cases of tumors (pathologically diagnosed as breast cancer and labeled as cancer), 91 cases of benign lesions (pathologically diagnosed as benign and labeled as benign), and 113 cases of healthy controls (labeled as normal). Raman spectra of the serum samples from each group were collected. To classify the normal, benign, and cancer sample groups, principal component analysis (PCA) combined with support vector machine (SVM) was used. The SVM model was evaluated using a cross-validation method. Results: The results of the study revealed significant differences in the mean Raman spectra of the serum samples between the normal and tumor/benign groups. Although the mean Raman spectra showed slight variations between the cancer and benign groups, the SVM model achieved a remarkable prediction accuracy of up to 98% for classifying cancer, benign, and normal groups. Discussion: In conclusion, this exploratory study has demonstrated the tremendous potential of general Raman spectroscopy as a clinical adjunctive diagnostic and rapid screening tool for breast cancer.

Research of integrated shimming Halbach magnet for High strength, compact Benchtop NMR device.

Due to production and assembly errors of magnets resulting in reduced magnetic field homogeneity, passive shimming (PS) is necessary when Halbach magnets are used in high-resolution Benchtop Nuclear Magnetic Resonance (BNMR) spectrometer. The conventional PS technique, which places independent PS devices inside the magnet aperture, is no longer applicable to small-aperture compact high-field-strength Halbach magnet studies. In this paper, based on spherical harmonic function expansion, we improve the magnetic field homogeneity by optimizing the moving step of the magnet moving arrays composed of Halbach magnets to generate the corresponding harmonic terms to compensate for the magnetic field. With this approach, the homogeneity of a 1 T Halbach magnet was improved from the original 3913 ppm to 8 ppm in a L10 mm × R2.5 mm of 0.64% copper sulfate doped water sample. This work explores the PS mechanism based on the movement of magnetic blocks, which can be applied in BNMR and other compact high-field strength high-homogeneity Halbach magnets application circumstances.

The Light- and Jasmonic Acid-Induced AaMYB108-like Positive Regulates the Initiation of Glandular Secretory Trichome in Artemisia annua L.

The plant Artemisia annua L. is famous for producing "artemisinin", which is an essential component in the treatment of malaria. The glandular secretory trichomes (GSTs) on the leaves of A. annua secrete and store artemisinin. Previous research has demonstrated that raising GST density can effectively raise artemisinin content. However, the molecular mechanism of GST initiation is not fully understood yet. In this study, we identified an MYB transcription factor, the AaMYB108-like, which is co-induced by light and jasmonic acid, and positively regulates glandular secretory trichome initiation in A. annua. Overexpression of the AaMYB108-like gene in A. annua increased GST density and enhanced the artemisinin content, whereas anti-sense of the AaMYB108-like gene resulted in the reduction in GST density and artemisinin content. Further experiments demonstrated that the AaMYB108-like gene could form a complex with AaHD8 to promote the expression of downstream AaHD1, resulting in the initiation of GST. Taken together, the AaMYB108-like gene is a positive regulator induced by light and jasmonic acid for GST initiation in A. annua.

Piriformospora indica alter root-associated microbiome structure to enhance Artemisia annua L. tolerance to arsenic.

Microorganisms in the rhizosphere are crucial allies for plant stress tolerance. Recent research suggests that by interacting with the rhizosphere microbiome, microorganisms can aid in the revegetation of soils contaminated with heavy metal(loid)s (HMs). However, it is unknown that how Piriformospora indica influences the rhizosphere microbiome to mitigate arsenic-toxicity in arsenic-enriched environments. Artemisia annua plants were grown in the presence or absence of P. indica and spiked with low (50) and high (150 µmol/L) concentrations of arsenic (As). After inoculation with P. indica, fresh weight increased by 37.7% and 10% in control and high concentration treated plants, respectively. Transmission electron microscopy showed that cellular organelles were severely damaged by As and even disappeared under high concentration. Furthermore, As was mostly accumulated by 5.9 and 18.1 mg/kg dry weight in the roots of inoculated plants treated with low and high concentrations of As, respectively. Additionally, 16 S and ITS rRNA gene sequencing were applied to analyze the rhizosphere microbial community structure of A. annua under different treatments. A significant difference was observed in microbial community structure under different treatments as revealed by non-metric multidimensional scaling ordination. The bacterial and fungal richness and diversity in the rhizosphere of inoculated plants were actively balanced and regulated by P. indica co-cultivation. Lysobacter and Steroidobacter were found to be the As-resistant bacterial genera. We conclude that P. indica inoculation could alter rhizosphere microecology, thereby mitigating As-toxicity without harming the environment.

JA-Regulated AaGSW1-AaYABBY5/AaWRKY9 Complex Regulates Artemisinin Biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene lactone obtained from Artemisia annua, is an essential therapeutic against malaria. YABBY family transcription factor AaYABBY5 is an activator of AaCYP71AV1 (cytochrome P450-dependent hydroxylase) and AaDBR2 (double-bond reductase 2); however, the protein-protein interactions of AaYABBY5, as well as the mechanism of its regulation, have not yet been elucidated. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (glandular trichome-specific WRKY1) and AaDBR2 (double-bond reductase 2). In this study, YABBY-WRKY interactions are revealed to indirectly regulate artemisinin production. AaYABBY5 significantly increased the activity of the luciferase (LUC) gene fused to the promoter of AaGSW1. Toward the molecular basis of this regulation, AaYABBY5 interaction with AaWRKY9 protein was found. The combined effectors AaYABBY5 + AaWRKY9 showed synergistic effects toward the activities of AaGSW1 and AaDBR2 promoters, respectively. In AaYABBY5 overexpression plants, the expression of GSW1 was found to be significantly increased when compared to that of AaYABBY5 antisense or control plants. In addition, AaGSW1 was identified as an upstream activator of AaYABBY5. Further, it was found that AaJAZ8, a transcriptional repressor of jasmonate signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and anti-AaJAZ8 in A. annua increased the activity of AaYABBY5 toward artemisinin biosynthesis. This current study provides the first indication of the molecular basis of regulation of artemisinin biosynthesis through YABBY-WRKY interactions, which are regulated through AaJAZ8. This knowledge presents AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis.

AaSEPALLATA1 integrates jasmonate and light-regulated glandular secretory trichome initiation in Artemisia annua.

Glandular secretory trichomes (GSTs) can secrete and store a variety of specific metabolites. By increasing GST density, valuable metabolites can be enhanced in terms of productivity. However, the comprehensive and detailed regulatory network of GST initiation still needs further investigation. By screening a complementary DNA library derived from young leaves of Artemisia annua, we identified a MADS-box transcription factor, AaSEPALLATA1 (AaSEP1), that positively regulates GST initiation. Overexpression of AaSEP1 in A. annua substantially increased GST density and artemisinin content. The HOMEODOMAIN PROTEIN 1 (AaHD1)-AaMYB16 regulatory network regulates GST initiation via the jasmonate (JA) signaling pathway. In this study, AaSEP1 enhanced the function of AaHD1 activation on downstream GST initiation gene GLANDULAR TRICHOME-SPECIFIC WRKY 2 (AaGSW2) through interaction with AaMYB16. Moreover, AaSEP1 interacted with the JA ZIM-domain 8 (AaJAZ8) and served as an important factor in JA-mediated GST initiation. We also found that AaSEP1 interacted with CONSTITUTIVE PHOTOMORPHOGENIC 1 (AaCOP1), a major repressor of light signaling. In this study, we identified a MADS-box transcription factor that is induced by JA and light signaling and that promotes the initiation of GST in A. annua.

AaWIN1, an AP2/ERF protein, positively regulates glandular secretory trichome initiation in Artemisia annua.

Exploring the genetic network of glandular trichomes and manipulating genes relevant to secondary metabolite biosynthesis are of great importance and value. Artemisinin, a key antimalarial drug ingredient, is synthesized and stored in glandular secretory trichomes (GSTs) in Artemisia annua. WIN/SHN proteins, a clade of AP2/ERF family, are known as regulators for cuticle biosynthesis. However, their function in glandular trichome development is less unknown. In this study, we identified a WIN/SHN gene from A. annua and named it as AaWIN1. AaWIN1 was predominantly expressed in buds, flowers and trichomes, and encoded a nuclear-localized protein. Overexpressing AaWIN1 in A. annua significantly increased the density of GST as well as the artemisinin content. Furthermore, AaGSW2 was reported to play an important role in promoting GST initiation, and the expression of AaGSW2 was induced in AaWIN1-overexpression lines. AaMIXTA1, a MYB protein positively regulating trichome initiation and cuticle biosynthesis, was confirmed to interact with AaWIN1. In addition, the ectopic expression of AaWIN1 resulted in slender and curled leaves, fewer trichomes, and rising expressions of cuticle biosynthesis genes in Arabidopsis thaliana. Taken together, based on phenotype observations, content measurements and gene expression detections, AaWIN1 was considered as a positive regulator for GST initiation in A. annua.

Theoretical foundation for designing multilayer Halbach array magnets for benchtop NMR and MRI.

Multilayer Halbach array magnets support portable NMR and MRI, but optimizing their design to maximize performance and minimize the use of expensive magnet materials is challenging. This is partly because our theoretical understanding of such arrays is incomplete and computationally intensive. Here we provide a theoretical description of the magnetic field distribution and we demonstrate that inhomogeneity is greatest along the z axis in multilayer Halbach array magnets. This allows the configuration of the multilayer Halbach array magnets to be optimized in a way that takes into account homogeneity, magnet volume, and magnetic flux density. At the same time, our description simplifies the design of multilayer array magnets, while accommodating the possibility of different outer radii, lengths for each layer array, or the presence of separation between the rings. We validated the theoretical description in simulations of a three-layer Halbach array magnet, then with a prototype three-layer 1-T Halbach array magnet. After adjusting the position of magnet blocks in the neighboring rings, we achieved homogeneity of 220 ppm for a standard 5 mm NMR tube while the inner diameter of the magnet is 20 mm. Our work provides a theoretical foundation for designing multilayer Halbach array magnets to maximize homogeneity and minimize the use of magnet materials.

A high-efficiency trichome collection system by laser capture microdissection.

Trichomes, which are classified as glandular or non-glandular, are hair-like epidermal structures that are present on aerial parts of most plant species. Glandular secretory trichomes (GSTs) have the capacity to secrete and store specialized metabolites, which are widely used as natural pesticides, food additives, fragrance ingredients or pharmaceuticals. Isolating individual trichomes is an essential way for identifying trichome-specific gene functions and discovering novel metabolites. However, the isolation of trichomes is difficult and time-consuming. Here, we report a method to isolate the GSTs from leaf epidermis dispense with fixation using laser capture microdissection (LCM). In this study, 150 GSTs were captured efficiently from Artemisia annua leaves and enriched for artemisinin measurement. UPLC analysis of microdissected samples indicated specific accumulation of secondary metabolites could be detected from a small number of GSTs. In addition, qRT-PCR revealed that the GST-specific structural genes involved in artemisinin biosynthesis pathway were highly expressed in GSTs. Taken together, we developed an efficient method to collect comparatively pure GSTs from unfixed leaved, so that the metabolites were relatively obtained intact. This method can be implemented in metabolomics research of purely specific plant cell populations and has the potential to discover novel secondary metabolites.

MADS-box gene AaSEP4 promotes artemisinin biosynthesis in Artemisia annua.

The plant Artemisia annua is well known for its production of artemisinin, a sesquiterpene lactone that is an effective antimalarial compound. Although remarkable progress has been made toward understanding artemisinin biosynthesis, the effect of MADS-box family transcription factors on artemisinin biosynthesis is still poorly understood. In this study, we identified a MADS transcription factor, AaSEP4, that was predominantly expressed in trichome. AaSEP4 acts as a nuclear-localized transcriptional activator activating the expression of AaGSW1 (GLANDULAR TRICHOME-SPECIFIC WRKY1). Dual-luciferase and Yeast one-hybrid assays revealed that AaSEP4 directly bound to the CArG motif in the promoter region of AaGSW1. Overexpression of AaSEP4 in A. annua significantly induced the expression of AaGSW1 and four artemisinin biosynthesis genes, including amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), double-bond reductase 2 (DBR2) and aldehyde dehydrogenase 1 (ALDH1). Furthermore, the results of high-performance liquid chromatography (HPLC) showed that the artemisinin content was significantly increased in the AaSEP4-overexpressed plants. In addition, RT-qPCR results showed that AaSEP4 was induced by methyl jasmonic acid (MeJA) treatment. Taken together, these results explicitly demonstrate that AaSEP4 is a positive regulator of artemisinin biosynthesis, which can be used in the development of high-artemisinin yielding A. annua varieties.

Engineering the expression of plant secondary metabolites-genistein and scutellarin through an efficient transient production platform in Nicotiana benthamiana L.

Plant natural products (PNPs) are active substances indispensable to human health with a wide range of medical and commercial applications. However, excessive population growth, overexploitation of natural resources, and expensive total chemical synthesis have led to recurrent supply shortages. Despite the fact that the microbial production platform solved these challenges, the platform still has drawbacks such as environmental pollution, high costs, and non-green production. In this study, an efficient platform for the production of PNPs based on the transient expression system of Nicotiana benthamiana L. combined with synthetic biology strategies was developed. Subsequently, the feasibility of the platform was verified by a simple "test unit." This platform was used to synthesize two high-value PNPs: genistein (5.51 nmol g-1 FW) and scutellarin (11.35 nmol g-1 FW). Importantly, this is the first report on the synthesis of scutellarin in heterologous plants. The platform presented here will possibly be adopted for the heterologous production of genistein and scutellarin in tobacco plants as a novel and sustainable production strategy.

Application of Surface-Enhanced Raman Spectroscopy in the Screening of Pulmonary Adenocarcinoma Nodules.

This study is aimed at evaluating the feasibility of a screening method for the pulmonary adenocarcinoma nodules through surface-enhanced Raman spectroscopy (SERS). Objective. Using SERS to measure serum from pulmonary nodules and healthy subjects, intraoperative biopsy pathological diagnosis was regarded as the gold standard for labeling serum samples. To explore the application value of SERS in the differential diagnosis of pulmonary adenocarcinoma nodules, benign nodules, and healthy, we build a machine learning model. Method. We collected 116 serum samples from patients. Radiographically confirmed nodules less than 3 cm in maximum diameter in all patients, including 58 cancer (pathologic diagnosis: adenocarcinoma nodules, labeled as cancer) patients, 58 pathologic diagnoses as benign nodule (labeled as benign) patients, and 63 healthy (labeled as normal) people from the clinical laboratory of Sichuan Cancer Hospital. Gold nanorods were employed as SERS substrates. Support vector machine (SVM) was used to classify the normal, benign, and cancer sample groups, and SVM model evaluated using cross-validation. Results. The average SERS spectra of serum were significantly different between the normal group and the cancer/benign group. While the average SERS spectra of the cancer group and the benign group differed slightly, for the cancer, benign, and normal groups, SVM models can predict with 93.33% accuracy. Conclusion. This exploratory study demonstrates that the SERS technique based on nanoparticles in conjunction with SVM has great potential as a clinical auxiliary diagnosis and screening for pulmonary adenocarcinoma nodules.

The truncated AaActin1 promoter is a candidate tool for metabolic engineering of artemisinin biosynthesis in Artemisia annua L.

Malaria is a devastating parasitic disease with high levels of morbidity and mortality worldwide. Artemisinin, the active substance against malaria, is a sesquiterpenoid produced by Artemisia annua. To improve artemisinin content in the native A. annua plants, considerable efforts have been attempted, with genetic transformation serving as an effective strategy. Although, the most frequently-used cauliflower mosaic virus (CaMV) 35S (CaMV35S) promoter has proved to be efficient in A. annua transgenic studies, it appears to show weak activity in peltate glandular secretory trichomes (GSTs) of A. annua plants. Here, we characterized the 1727 bp fragment upstream from the translation start codon (ATG) of AaActin1, however, found it was inactive in tobacco. After removal of the 5' intron, the truncated AaActin1 promoter (tpACT) showed 69% and 50% activity of CaMV35S promoter in transiently transformed tobacco and stably transformed A. annua, respectively. ß-glucuronidase (GUS) staining analysis showed that the tpACT promoter was capable of directing the constant expression of a foreign gene in peltate GSTs of transgenic A. annua, representing higher activity than CaMV35S promoter. Collectively, our study provided a novel promoter available for metabolic engineering of artemisinin biosynthesis in A. annua.

A passive shimming method for Halbach magnet based on magnetic sheet arrays.

Halbach magnet has great potential in nuclear magnetic resonance device, where the field homogeneity requirement puts heavy demands on high efficiencypassive shimming (PS) technique. Conventional PS involves a tedious iteration process of the magnetic block/sheet number and positions optimization. In this paper, we propose a PS method based on magnetic sheet arrays (MSAs) targeting at spherical harmonic basis up to the 3rd order including dedicated composition of Y(4Z2-X2-Y2), Z3 and X(4Z2-X2-Y2) (n = 3, m = -1,0,1) with cross terms to implement structural field compensation. With this approach, the homogeneity of a 0.5 T Halbach magnet was improved from the original 811 ppm to 4.7 ppm in a L15 mm × R2.5 mm water sample. Rough shimming in another 0.93 T Halbach magnet also improved the homogeneity from 1103 ppm to 125 ppm in R2.5 mm sphere. This work provides a flexible, convenient PS method based on MSAs for compact Halbach magnet, which can be applied in NMR spectrometers and other high homogeneity application circumstances.

Switching-behavior improvement in HfO2/ZnO bilayer memory devices by tailoring of interfacial and microstructural characteristics.

We investigated the effect of top contact interface and microstructural characteristics of the insulating layers on resistive switching behaviors by fabricating and characterizing the HfO2/ZnO bilayer heterostructures. Different thickness of ZnO underlying layer and different deposition temperatures of the upper HfO2layer were designed to analyze the intrinsic contribution of the crystalline microstructure of the insulating bilayer. Pt and Ti top electrodes were used to demonstrate the extrinsic contribution of the interface configuration. It was observed that all devices show bipolar RS characteristics. Unlike the device composed of Pt/HfO2/ZnO/Pt that exhibit an abrupt switching, a gradually continuous switching in the reset process was identified in the device composed of Ti/HfO2/ZnO/Pt. Interfacial charge migration process/characteristic plays a key role in the RS process as well as its conduction mechanism. The RS performance of the former is significantly better than that of the latter, including much lower reset voltage, two orders of magnitude larger OFF/ON ratio and HRS resistance. In addition, as compared to the intrinsic contribution arising from the microstructure of the HfO2/ZnO bilayer to the RS performances and current transport mechanism, the extrinsic effect contributed from the electrode characteristics (and its interface) is dominant.

Differential Graphical Games for Constrained Autonomous Vehicles Based on Viability Theory.

This article proposes an optimal-distributed control protocol for multivehicle systems with an unknown switching communication graph. The optimal-distributed control problem is formulated to differential graphical games, and the Pareto optimum to multiplayer games is sought based on the viability theory and reinforcement learning techniques. The viability theory characterizes the controllability of a wide range of constrained nonlinear systems; and the viability kernel and the capture basin are the pillars of the viability theory. The capture basin is the set of all initial states, in which there exist control strategies that enable the states to reach the target in finite time while remaining inside a set before reaching the target. In this regard, the feasible learning region is characterized by the reinforcement learner. In addition, the approximation of the capture basin provides the learner with prior knowledge. Unlike the existing works that employ the viability theory to solve control problems with only one agent and differential games with only two players, the viability theory, in this article, is utilized to solve multiagent control problems and multiplayer differential games. The distributed control law is composed of two parts: 1) the approximation of the capture basin and 2) reinforcement learning, which are computed offline and online, respectively. The convergence properties of the parameters' estimation errors in reinforcement learning are proved, and the convergence of the control policy to the Pareto optimum of the differential graphical game is discussed. The guaranteed approximation results of the capture basin are provided and the simulation results of the differential graphical game are provided for multivehicle systems with the proposed distributed control policy.

AaWRKY17, a positive regulator of artemisinin biosynthesis, is involved in resistance to Pseudomonas syringae in Artemisia annua.

Artemisia annua, a traditional Chinese medicinal plant, remains the only plant source for artemisinin production, yet few genes have been identified to be involved in both the response to biotic stresses, such as pathogens, and artemisinin biosynthesis. Here, we isolated and identified the WRKY transcription factor (TF) AaWRKY17, which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A. annua. Yeast one-hybrid (Y1H), dual-luciferase (dual-LUC), and electrophoretic mobility shift assay (EMSA) results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase (ADS) and promoted its expression. Real-time quantitative PCR (RT-qPCR) analysis revealed that the transcript levels of two defense marker genes, Pathogenesis-Related 5 (PR5) and NDR1/HIN1-LIKE 10 (NHL10), were greatly increased in AaWRKY17-overexpressing transgenic A. annua plants. Additionally, overexpression of AaWRKY17 in A. annua resulted in decreased susceptibility to P. syringae. These results indicated that AaWRKY17 acted as a positive regulator in response to P. syringae infection. Together, our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A. annua.