Identification of Molecular Profile of Cell Membrane via Magnetic Plasmonic Nanoprobe.

Cell membranes are primarily composed of lipids, membrane proteins, and carbohydrates, and the related studies of membrane components and structures at different stages of disease development, especially membrane proteins, are of great significance. Here, we investigate the chemical signature profiles of cell membranes as biomarkers for cancer cells via label-free surface-enhanced Raman scattering (SERS). A magnetic plasmonic nanoprobe was proposed by decorating magnetic beads with silver nanoparticles, allowing self-driven cell membrane-targeted accumulation within 5 min. SERS profiles of three types of breast cells were achieved under the plasmon enhancement effect of these nanoprobes. Membrane fingerprint spectra from breast cell lines were further classified with the convolutional neural network model, which perfectly distinguished between two breast cancer subtypes. We further tested various clinical samples using this method and obtained fingerprint spectra from primary cells and frozen slices. This study presents a practical, user-friendly approach for label-free and in situ analysis of cell membranes, which can work for early tumor screening and treatment assessment for tumors reliant on the Molecular profiles of cell membranes. Additionally, this method can be applied universally to explore cell membrane components of other cells, thus assisting Human Cell Atlas.

Single-Cell Analysis and Classification according to Multiplexed Proteins via Microdroplet-Based Self-Driven Magnetic Surface-Enhanced Raman Spectroscopy Platforms Assisted with Machine Learning Algorithms.

A microdroplet-based surface-enhanced Raman spectroscopy (microdroplet SERS) platform was constructed to envelop individual cells in microdroplets, followed by the SERS detection of their extracellular vesicle-proteins (EV-proteins) via the in-drop immunoassays by use of immunomagnetic beads (iMBs) and immuno-SERS tags (iSERS tags). A unique phenomenon is found that iMBs can start a spontaneous reorientation on the probed cell surface based on the electrostatic force-driven interfacial aggregation effect, which leads EV-proteins and iSERS tags to be gathered from a liquid phase to a cell membrane interface and significantly improves SERS sensitivity to the single-cell analysis level due to the formation of numbers of SERS hotspots. Three EV-proteins from two breast cancer cell lines were collected and further analyzed by machine learning algorithmic tools, which will be helpful for a deeper understanding of breast cancer subtypes from the view of EV-proteins.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

A SERS/fluorescence dual-mode immuno-nanoprobe for investigating two anti-diabetic drugs on EGFR expressions.

A surface-enhanced Raman scattering (SERS)/fluorescence dual-mode nanoprobe was proposed to assess anti-diabetic drug actions from the expression level of the epidermal growth factor receptor (EGFR), which is a significant biomarker of breast cancers. The nanoprobe has a raspberry shape, prepared by coating a dye-doped silica nanosphere with a mass of SERS tags, which gives high gains in fluorescence imaging and SERS measurement. The in situ detection of EGFR on the cell membrane surfaces after drug actions was achieved by using this nanoprobe, and the detection results agree with the enzyme-linked immunosorbent assay (ELISA) kit. Our study suggests that rosiglitazone hydrochloride (RH) may be a potential drug for diabetic patients with breast cancer, while the anti-cancer effect of metformin hydrochloride (MH) is debatable since MH slightly promotes the EGFR expression of MCF-7 cells in this study. This sensing platform endows more feasibility for highly sensitive and accurate feedback of pesticide effects at the membrane protein level.

High-quality chromosome-scale de novo assembly of the Paspalum notatum 'Flugge' genome.

BACKGROUND: Paspalum notatum 'Flugge' is a diploid with 20 chromosomes (2n = 20) multi-purpose subtropical herb native to South America and has a high ecological significance. It is currently widely planted in tropical and subtropical regions. Despite the gene pool of P. notatum 'Flugge' being unearthed to a large extent in the past decade, no details about the genomic information of relevant species in Paspalum have been reported. In this study, the complete genome information of P. notatum was established and annotated through sequencing and de novo assembly of its genome. RESULTS: The latest PacBio third-generation HiFi assembly and sequencing revealed that the genome size of P. notatum 'Flugge' is 541 M. The assembly result is the higher index among the genomes of the gramineous family published so far, with a contig N50 = 52Mbp, scaffold N50 = 49Mbp, and BUSCOs = 98.1%, accounting for 98.5% of the estimated genome. Genome annotation revealed 36,511 high-confidence gene models, thus providing an important resource for future molecular breeding and evolutionary research. A comparison of the genome annotation results of P. notatum 'Flugge' with other closely related species revealed that it had a close relationship with Zea mays but not close compared to Brachypodium distachyon, Setaria viridis, Oryza sativa, Puccinellia tenuiflora, Echinochloa crusgalli. An analysis of the expansion and contraction of gene families suggested that P. notatum 'Flugge' contains gene families associated with environmental resistance, increased reproductive ability, and molecular evolution, which explained its excellent agronomic traits. CONCLUSION: This study is the first to report the high-quality chromosome-scale-based genome of P. notatum 'Flugge' assembled using the latest PacBio third-generation HiFi sequencing reads. The study provides an excellent genetic resource bank for gramineous crops and invaluable perspectives regarding the evolution of gramineous plants.

Microfluidic Droplet-SERS Platform for Single-Cell Cytokine Analysis via a Cell Surface Bioconjugation Strategy.

A microfluidic-based surface-enhanced Raman scattering (SERS) platform for analyzing cytokines secreted by single cells is reported based on the elaborate bioconjugation of the immuno-sandwich complex on the probed cell surface. This platform integrates the dual functions of microfluidic droplet separation of single cells and SERS measurement. Two immune nanoprobes (capture probe and SERS probe) are introduced into a microfluidic droplet along with a single cell. They were anchored to the cell membrane protein surface by capturing secreted cytokines to form an immune sandwich structure, realizing the enrichment effect of cytokines above the cell membrane surface and the amplification effect of SERS detection probes. This single-cell analytical platform was applied to track specific cell-secreted vascular endothelial growth factor (VEGF) of different cell lines (MCF-7, SGC, and T24), and highly sensitive detection of VEGF was achieved. Chemometric methods (principal component analysis and t-distributed stochastic neighbor embedding) were adopted for the SERS data analysis, and the support vector machine (SVM) discriminant model was established to test the data. These chemometric methods successfully identify significant differences in the secreting ability of cytokines among three kinds of cancer cell lines, revealing cell heterogeneity. In addition, the behavior of single cells secreting VEGF was monitored time-dependently and was shown to increase with time. This work demonstrates the importance of tracking specific cells secreting cytokines based on the cell surface bioconjugation strategy. Our developed platform provides guidelines for using the single-cell exocytosis factors as biomarkers to assess the early diagnosis of cancer and provide physiological cues for learning single-cell secretions.

Single-Cell VEGF Analysis by Fluorescence Imaging-Microfluidic Droplet Platform: An Immunosandwich Strategy on the Cell Surface.

Despite recent advances in single-cell analysis techniques, the ability of single-cell analysis platforms to track specific cells that secreted cytokines remains limited. Here, we report a microfluidic droplet-based fluorescence imaging platform that can analyze single cell-secreted vascular endothelial growth factor (VEGF), an important regulator of physiological and pathological angiogenesis, to explore cellular physiological clues at the single-cell level. Two kinds of silica nanoparticle (NP)-based immunoprobes were developed, and they were bioconjugated to the membrane proteins of the probed cell surface via the bridging of secreted VEGF. Thus, an immunosandwich assay was built above the probed cell via fluorescence imaging analysis of each cell in isolated droplets. This analytical platform was used to compare the single-cell VEGF secretion ability of three cell lines (MCF-7, HeLa, and H8), which experimentally demonstrates the cellular heterogeneity of cells in secreting cytokines. The uniqueness of this method is that the single-cell assay is carried out above the cell of interest, and no additional carriers (beads or reporter cells) for capturing analytes are needed, which dramatically improves the availability of microdroplets. This single-cell analytical platform can be applied for determining other secreted cytokines at the single-cell level by changing other immune pairs, which will be an available tool for exploring single-cell metabonomics.

First report of alfalfa root rot caused by Trichothecium roseum in China.

Alfalfa (Medicago sativa L.) is perennial leguminous forage, which is cultivated throughout the world due to its high yield, high quality, satisfactory palatability, and wide adaptability. With the increase of planting area in China, root diseases caused by Fusarium spp., Sclerotium rolfsii, Phytophthora spp. (Yang et al. 2022), and new pathogens have been found that reduce the yield and quality of alfalfa and cause economic losses (Li at al. 2019). In 2021, an alfalfa disease occurred under conditions of high temperature and high humidity at the Jiaozhou Experimental Base of Qingdao Agricultural University (Jiaozhou Modern Agricultural Science and Technology Demonstration Park, 36.33°N 120.40°E, Qingdao, Shandong, China), and about 2 ha of alfalfa were infected. The disease affected up to 35% of the plants and caused grass spots. Infected plants developed black-brown lesions with irregular shapes on roots with yellowing of the foliage; the leaves of the whole plant turned yellow. In the late stage of the disease, defoliation occurred and the plants stopped growing, wilted and died. Ten infected plants with typical symptom were collected for isolation and identification of pathogen. The infected roots were cut into 3-5 mm2 sections and then soaked in 75% ethanol for 30 s, followed by a 3-minute immersion in 2% sodium hypochlorite for surface sterilization. Next, the tissues were rinsed in sterile water five times and then placed on potato dextrose agar (PDA) medium. After three subcultures and subsequent single spore isolation, one representative strain named as DC1 was isolated from the infected roots. Based on morphological observation, the colony of DC1 was flat, granular, and powdery in appearance. Four days after inoculation on PDA medium, the size of the colony were 2.1-2.6 cm. After 8 to 20 days, the colonies were initially white and gradually change a light pink to peach color. The conidia are two-celled (Hamid et al. 2014), elliptic to pear-shaped, colorless or translucent, smooth to slightly rough with thick walls. The size of conidia ranged from 11.3 to 23.5 µm long × 6.1 to 12.7 µm wide (n =30). For the identification, the rDNA--ITS gene of the fungus was amplified using the primers ITS1/ITS4 (White et al.1990), and the EF1α gene was amplified using primers EF1-983F/EF1-2218R (Rehner and Buckley 2005). Then the PCR amplicons were cloned into the pCE2 TA/Blunt-Zero vector. The results of the rDNA-ITS (OM049197.1, 515 bp) and EF1α (OM069381.1, 926 bp) sequences were deposited in GenBank. DNA analysis showed that the two sequences were 100% similar to the rDNA-ITS sequence (MN882763.1) and EF1α sequence (DQ676610.1) of Trichothecium roseum, respectively. A pathogenicity test was done by placing one piece (0.5 cm in diameter) of fungal culture (PDA plug) 1cm below the crown of 40-day-old healthy alfalfa (cv. Zhongmu No.3) plants, 3 replicates and 20 plants in each replicate. PDA plug without the pathogen were used for control. All plants were cultivated in a growth chamber at 25±1°C with a light cycle of 15 h (90% relative humidity). After 18 days, the roots of inoculated plants had dark brown lesions and the leaves of their plants turn yellow, while those control plants had no symptoms. To fulfilling Koch's postulates, the same pathogen was re-isolated from necrotic root tissue of inoculated plants and confirmed by morphology and the rDNA-ITS and EF1-α sequences. Based on disease symptoms, morphological characteristics DNA sequences and pathogenicity, the pathogen of alfalfa disease in Jiaozhou Experimental Base of Qingdao Agricultural University was identified as T. roseum. To our knowledge, this is first report of T. roseum causing alfalfa root rot. The newly emerging disease may pose threat to alfalfa production of central and southern China in future.

Identification of a gene responsible for seedpod spine formation and other phenotypic alterations using whole-genome sequencing analysis in Medicago truncatula.

In nature, some plant species produce seedpods with spines, which is an adaptive biological trait for protecting the seed and helping seed dispersal. However, the molecular mechanism of spine formation is still unclear. While conducting routine tissue culture and transformation in the model legume Medicago truncatula, we identified a smooth seedpod (ssp1) mutant with a suite of other phenotypic changes. Preliminary analysis showed that the mutation was derived from the tissue culture process. Genetic segregation analysis suggested that ssp1 is a recessive mutant. By combining whole-genome sequencing and bioinformatics analysis, we found that the mutant phenotype was caused by a single nucleotide polymorphism and a 30 bp deletion in the gene locus Medtr4g039430, named SSP1. Complementation of the M. truncatula ssp1 and Arabidopsis twd1 mutants showed complete restoration, indicating that SSP1 is an ortholog of Arabidopsis TWD1 which encodes an immunophilin-like FK506-binding protein 42. The formation of spines on seedpods is associated with auxin transport. The method used in this study offers an effective way for detecting genes responsible for somaclonal variations. The results demonstrate, for the first time, that SSP1 plays a crucial role in the determination of spine formation on seedpods.

Southern Blight on Medicago sativa Caused by Sclerotium rolfsii in North China.

Alfalfa (Medicago sativa L.) is one of the most important perennial leguminous forages in many countries, known by its high feed value and yield potential. With the increasing demand for feed, alfalfa has been planted all over China. However, an increasingly serious alfalfa disease was observed and may restrict the development of the alfalfa industry in North China. In August 2019, an emerging alfalfa disease with symptoms resembling southern blight was observed in Jiaozhou experimental base (Jiaozhou Modern Agricultural Science and Technology Demonstration Park) of Qingdao Agricultural University (Qindao, Shandong province, China). The infected plants showed dark brown lesions on the stems and yellowing and wilting of the leaves. The pathogen produced white fluffy mycelia, and later sclerotia on stems and roots; the disease affected up to 25% of the plants and causes bare spots filled with weeds (Figure S1). Typical symptomatic tissues were brought back to the laboratory for pathogen isolation and identification. Fragments (3-5mm2) of root tissues were excised from lesions on the symptomatic roots and their surfaces were disinfested by sequential dipping in 70% ethanol for 30 s and in 2% NaClO for 3 min, then the fragments were rinsed in sterile water five times and cultured on potato dextrose (PDA) medium amended with streptomycin sulfate (0.1mg/mL). Cultures were incubated at 28°C in the dark and purified in PDA medium for three times. A representative strain (coded as CZL1) was isolated from the root rot of the diseased plant. After four days incubation on PDA, CZL1 formed white fluffy aerial mycelium 5.6-6 cm in diameter typical of S. rolfsii. After 15 to 20 days, abundant round sclerotia approximately1-3 mm in diameter were produced on the surface of the culture (Figure S2). The sclerotia were white at first and then gradually turned dark brown. To confirm the identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA region of the fungus was amplified using the primers ITS1/ITS4 (White et al.1990), and the elongation factor-1a gene (EF1a) was amplified using primers EF1-983F/EF1-2218R (Rehner and Buckley 2005). Then the PCR amplicons were cloned into the pCE2 TA/Blunt-Zero vector. The isolate was determined to contain two distinct sequence types for each gene. The results of ITS (MT812692, MT812693) and EF1a (MT846496 and MT846497) sequences were deposited in GenBank. DNA analysis revealed that the two ITS sequences were more than 99% identical to Athelia rolfsii (MN872304) in the NCBI GenBank database, and two EF1a sequences were 99% identical to the A. rolfsii EF1a sequence MN702789 and KP982854. To fulfill Koch's postulates, infected sorghum grain was placed near the roots of 15 40-day-old healthy alfalfa seedlings split into 3 pots with the same number of seedlings receiving a control treatment of sterilized sorghum grain. All plants were incubated in growth chamber at 24±1°C with 14-h-photoperiod (85% relative humidity). After 10-15 days, blight symptoms identical to those in the field were observed on inoculated plants, whereas those control plants were symptomless (Figure S2). S. rolfsii was successfully re-isolated from the inoculated plants and molecularly characterized as described above. Based on disease symptoms, fungal colonies, the ITS and EF1a sequence, and pathogenicity to the host, this fungus was identified as S. rolfsii (teleomorph Athelia rolfsii). To our knowledge, this is the first report of S. rolfsii as the causal agent of southern blight of alfalfa in North China, and it is also the first report of southern blight on alfalfa caused by S. rolfsii in China since 1996 observed in Guizhou province (Mo and Luo 1996).

De novo hydroponics system efficiency for the cuttings of alfalfa (Medicago sativa L.).

The legume plant alfalfa (Medicago sativa L.) is a widely cultivated perennial forage due to its high protein content, palatability, and strong adaptability to diverse agro-ecological zones. Alfalfa is a self-incompatible cross-pollinated autotetraploid species with tetrasomic inheritance. Therefore, maintaining excellent traits through seed reproduction is a prime challenge in alfalfa. However, the cutting propagation technology could enable consistent multiplication of quality plants that are genetically identical to the parent plant. The current study aimed to develop a simple, cost-effective, reproducible, and efficient hydroponic cutting method to preserve alfalfa plants and for molecular research. In this study, alfalfa landrace 'Wudi' was grown in hydroponics for 30 days and used as source material for cuttings. The top, middle and bottom sections of its stem were used as cuttings. The rooting rate, root length, and stem height of the different stem sections were compared to determine the best segment for alfalfa propagation in four nutrient treatments (HM, HM + 1/500H, HM + 1/1000H and d HM + 1/2000H). After 21 days of culture, the rooting rates of all the three stem types under four cutting nutrient solutions were above 78%. The rooting rate of the middle and bottom parts in HM + 1/1000 H and HM + 1/2000 H nutrient solutions reached more than 93%, with a higher health survey score (> 4.70). In conclusion, this study developed a de novo cutting propagation method that can be used to conserve and propagate germplasm in breeding programs and research. This method is a new report on the cutting propagation of alfalfa by hydroponics, which could supplement the existing cutting propagation methods.

Revealing Mitochondrial Microenvironmental Evolution Triggered by Photodynamic Therapy.

Mitochondrion is one of the most important organelles and becomes a target in many cancer therapeutic strategies. Mitochondrial microenvironments in response to therapeutic methods are the key to understand therapeutic mechanisms. However, they are almost rarely studied. Herein, the mitochondrial microenvironments, including mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) after different photodynamic therapy (PDT) dosages, were monitored by fluorescent imaging and compared among three cell lines (HepG2, MCF-7, and LO2). Furthermore, the fluctuations of intramitochondrial pHs were revealed via a plasmonic mitochondrion-targeting surface-enhanced Raman scattering (SERS) pH nanosensor. Results indicate that the MMP decreases gradually with the ROS generation and the cancerous cells exhibit less response to excess ROS relative to normal cells. On the other hand, the pH value in the mitochondria decreases initially and then increases when the amount of ROS increases. The LO2 cell is preliminarily evidenced to have a higher self-adjustment ability due to its better tolerance to differential intra/extracellular pHs. This study may provide a basis for an in-depth understanding of the mechanisms of the mitochondrial targeting-based PDT therapeutic processes. It is also helpful for more accurate and useful diagnosis according to intramitochondrial microenvironments and improvement on therapy efficiency of cancers.

Plasmon-Enhanced Four-Wave Mixing Imaging for Microdroplet-Based Single-Cell Analysis.

A high-throughput single-cell analytical technique based on the microdroplet array integrated with the plasmon-enhanced-four-wave mixing (PE-FWM) imaging was developed, which is applicable for the highly sensitive and automatic assessment of the surface receptors of cells. The metal nanoprobes were prepared by simply decorating metal nanoparticles with capturing molecules (antibody or molecules with surface identification function). Owing to the multifrequency selection of lasers via resonating their plasmonic bands, these metal nanoprobes are highly recognizable under the FWM imaging and display high photostability above fluorescent dyes. This PE-FWM imaging technique shows superior to dark-field imaging due to almost no interference from off-resonant species and exhibits the antifade feature that is suitable for long-period cell monitoring. The automated processing of images is available for the analysis of cell heterogeneity according to the cell surface receptors. Emerging applications such as single-cell analysis, bioimaging, metabolite, and drug tracing offer many biological and medical possibilities with broad application prospects.

Improving the genome editing efficiency of CRISPR/Cas9 in Arabidopsis and Medicago truncatula.

MAIN CONCLUSION: An improved CRISPR/Cas9 system with the Arabidopsis UBQ10 promoter-driven Cas9 exhibits consistently high mutation efficiency in Arabidopsis and M. truncatula. CRISPR/Cas9 is a powerful genome editing technology that has been applied in several crop species for trait improvement due to its simplicity, versatility, and specificity. However, the mutation efficiency of CRISPR/Cas9 in Arabidopsis and M. truncatula (Mt) is still challenging and inconsistent. To analyze the functionality of the CRISPR/Cas9 system in two model dicot species, four different promoter-driven Cas9 systems to target phytoene desaturase (PDS) genes were designed. Agrobacterium-mediated transformation was used for the delivery of constructed vectors to host plants. Phenotypic and genotypic analyses revealed that the Arabidopsis UBQ10 promoter-driven Cas9 significantly improves the mutation efficiency to 95% in Arabidopsis and 70% in M. truncatula. Moreover, the UBQ10-Cas9 system yielded 11% homozygous mutants in the T1 generation in Arabidopsis. Sequencing analyses of mutation events indicated that single-nucleotide insertions are the most frequent events in Arabidopsis, whereas multi-nucleotide deletions are dominant in bi-allelic and mono-allelic homozygous mutants in M. truncatula. Taken together, the UBQ10 promoter facilitates the best improvement in the CRISPR/Cas9 efficiency in PDS gene editing, followed by the EC1.2 promoter. Consistently, the improved UBQ10-Cas9 vector highly enhanced the mutation efficiency by four-fold over the commonly used 35S promoter in both dicot species.

Distinguishing cancer cell lines at a single living cell level via detection of sialic acid by dual-channel plasmonic imaging and by using a SERS-microfluidic droplet platform.

A high-throughput, dual-channel single cell analytical method is described for the detection of sialic acid (SA) on single cell based on the use of microfluidic droplets integrated with plasmonic imaging and surface-enhanced Raman spectroscopy (SERS) with the assistance of a multifunctional metal nanoparticle-based probe. The multifunctional plasmonic nanoprobe was prepared by modifying silver nanoparticles (AgNPs) with 4-mercaptophenylboronic acid (MPBA) that both warrants SA recognition and acts as a Raman reporter. This nanoprobe is a high-contrast indicator under bright field imaging due to the strong energy loss feature of AgNPs, and also owns possesses a strong SERS enhancement capability toward MPBA. Cells incubated with the plasmonic nanoprobes were isolated in water-in-oil droplets and then were re-dispersed in a chamber array chip. High-precision profiles of SA on a single cell in one droplet were obtained by the bright field imaging and image processing. The SA expression levels on different cell lines (MCF-7, HepG2, SGC and BNL.CL2) traced by SERS spectroscopy were compared. The statistical data among different cell lines confirm that the SA expression levels on cancer cells are much higher than that on normal cells. Single cell analysis further revealed that the cell-to-cell variations are more obvious in cancer cell lines. This study provides a valuable tool for understanding glycan-related biochemical processes. Graphical abstract A high-throughput, dual-channel microfluidic droplet platform succeeded in distinguishing different cancer cell lines at single living cell level integrated with plasmonic imaging and surface-enhanced Raman spectroscopy with assistance of a multifunctional metal nanoparticle-based probe.

Isolation and functional characterization of salt-stress induced RCI2-like genes from Medicago sativa and Medicago truncatula.

Salt stress is one of the most significant adverse abiotic factors, causing crop failure worldwide. So far, a number of salt stress-induced genes, and genes improving salt tolerance have been characterized in a range of plants. Here, we report the isolation and characterization of a salt stress-induced Medicago sativa (alfalfa) gene (MsRCI2A), which showed a high similarity to the yeast plasma membrane protein 3 gene (PMP3) and Arabidopsis RCI2A. The sequence comparisons revealed that five genes of MtRCI2(A-E) showed a high similarity to MsRCI2A in the Medicago truncatula genome. MsRCI2A and MtRCI2(A-E) encode small, highly hydrophobic proteins containing two putative transmembrane domains, predominantly localized in the plasma membrane. The transcript analysis results suggest that MsRCI2A and MtRCI2(A-D) genes are highly induced by salt stress. The expression of MsRCI2A and MtRCI2(A-C) in yeast mutants lacking the PMP3 gene can functionally complement the salt sensitivity phenotype resulting from PMP3 deletion. Overexpression of MsRCI2A in Arabidopsis plants showed improved salt tolerance suggesting the important role of MsRCI2A in salt stress tolerance in alfalfa.

In-droplet multiplex immunoassays for hypoxia-induced single-cell cytokines.

Cytokine expression is an important biomarker in understanding hypoxia microenvironments in tumor growth and metastasis. In-droplet-based immunoassays performed above the target cell membrane were employed to track the cytokines of single cells with the aid of three types of immuno-nanoprobes (one capture nanoprobe and two reporter nanoprobes). Single cells and nanoprobes were co-packaged in water-in-oil microdroplets (about 100 µm in diameter) using a cross-shaped microfluidic chip. In each droplet, capture nanoprobes would be first fixed to the cell surface by linking to membrane proteins that have been streptavidinized. Then, the capture nanoprobes can collect cell-secreted cytokines (VEGF and IL-8) by the antibodies, followed by two reporter nanoprobes that emit distinguishable fluorescence. Fluorescence imaging was utilized to record the signal outputs of two reporter probes, which reflect cytokine expressions secreted by a single tumor cell. The cytokine levels at different degrees of hypoxia induction were assessed. Multiple chemometric methods were adopted to distinguish differences in the secretion of two cytokines and the results demonstrated a positive correlation. This study developed an in-droplet, dual-target, simultaneous biosensing strategy for a single cell, which is helpful for understanding the impacts of hypoxia microenvironments on cell cytokines that are vital for assessing early cancer diagnosis and prognosis.

Mutating alfalfa COUMARATE 3-HYDROXYLASE using multiplex CRISPR/Cas9 leads to reduced lignin deposition and improved forage quality.

Alfalfa (Medicago sativa L.) forage quality is adversely affected by lignin deposition in cell walls at advanced maturity stages. Reducing lignin content through RNA interference or antisense approaches has been shown to improve alfalfa forage quality and digestibility. We employed a multiplex CRISPR/Cas9-mediated gene-editing system to reduce lignin content and alter lignin composition in alfalfa by targeting the COUMARATE 3-HYDROXYLASE (MsC3H) gene, which encodes a key enzyme in lignin biosynthesis. Four guide RNAs (gRNAs) targeting the first exon of MsC3H were designed and clustered into a tRNA-gRNA polycistronic system and introduced into tetraploid alfalfa via Agrobacterium-mediated transformation. Out of 130 transgenic lines, at least 73 lines were confirmed to contain gene-editing events in one or more alleles of MsC3H. Fifty-five lines were selected for lignin content/composition analysis. Amongst these lines, three independent tetra-allelic homozygous lines (Msc3h-013, Msc3h-121, and Msc3h-158) with different mutation events in MsC3H were characterized in detail. Homozygous mutation of MsC3H in these three lines significantly reduced the lignin content and altered lignin composition in stems. Moreover, these lines had significantly lower levels of acid detergent fiber and neutral detergent fiber as well as higher levels of total digestible nutrients, relative feed values, and in vitro true dry matter digestibility. Taken together, these results showed that CRISPR/Cas9-mediated editing of MsC3H successfully reduced shoot lignin content, improved digestibility, and nutritional values without sacrificing plant growth and biomass yield. These lines could be used in alfalfa breeding programs to generate elite transgene-free alfalfa cultivars with reduced lignin and improved forage quality.

Design of Low-RCS Broadband High-Gain Antennas Based on Transmission Array Metasurface.

In this paper, a low-RCS broadband high-gain antenna based on metasurface transmission array is proposed, consisting of two parts: a metasurface transmission array and a feed antenna. When designing the metasurface transmission array, the phase compensation method is used to achieve the beam convergence effect of metasurface in the broadband. By designing the elements and using the checkerboard arrangement, the RCS of the incident wave with fixed polarization can be reduced more than 10 dB at X band or Ku band. The feed antenna is designed as a microstrip magnetic and electric dipole antenna, which has the characteristics of small structure and wide impedance bandwidth. An antenna that can reduce RCS by more than 10 dB in Ku band is simulated and measured. The measurement and simulation results show that the -10 dB operating bandwidth of the high-gain antenna designed in this paper is 6.7~13.5 GHz, and the relative bandwidth is 67%. The designed metasurface can effectively improve the gain of the antenna in the operating frequency band. In this way, the design of high-gain antenna is realized, and the antenna has an obvious RCS reduction effect on the vertically incident y-polarized wave in the whole Ku band. The method to design an antenna in this paper realizes the regulation of radiation and scattering at the same time, which has important reference significance for expanding the function of transmission array antennae and has great application value.

Fluorescent Imaging Probe Targeting Mitochondria Based on Supramolecular Host-Guest Assembly and Disassembly.

Fluorescent dyes and probes play an indispensable role in bioimaging. The mitochondrion is one of the crucial organelles which takes charge of energy production and is the primary site of aerobic respiration in the cell. To illuminate mitochondria, a series of supramolecular fluorescent imaging probes were developed based on the host-guest assembly of 1,4-bis[2-(4-pyridyl)ethenyl]-benzene (BPEB) derivatives and cucurbituril[6] (CB[6]). These host-guest conjugates can be efficiently internalized into cells due to their water solubility and target mitochondria according to their positive charges. In response to the intracellular microenvironments, these conjugates start dynamic disassembly. The released BPEBs show a highly hydrophobic feature, which can crystallize to form fluorescent solids that illuminate the mitochondria. The intracellular disassembly of the host-guest probes was evidenced by fluorescence lifetime imaging in situ. These smart mitochondrion-targeting fluorescent imaging probes can be available to investigate the structures and functions of mitochondria, which are of great significance in the intracellular dynamic transformation of supramolecular assemblies.