Phosphorylation-amplified synchronized droplet microfluidics sensitizes bacterial growth kinetic real-time monitoring.

The necessity for rapid and accurate bacterial growth monitoring is imperative across various domains, including healthcare and environmental safety. We introduce the self-synchronized droplet-amplified electrical screening cytometry (SYNC) system, a novel meld of droplet microfluidics and electrochemical amplification tailored for precise bacterial growth kinetic monitoring. SYNC encapsulates single bacteria in picolitre droplets, enabling real-time, fluorescence-free electrochemical monitoring. A specially devised phosphorylation-amplified culture medium translates bacterial metabolic activity into discernible electrical impedance changes. The dual-channel design and a rail-based structure in SYNC facilitate parallel screening and self-synchronization of droplets, addressing the limitations of conventional impedance cytometry. SYNC showcases a 5-fold enhancement in detection sensitivity and reduces 50% of the detection time compared to traditional approaches. Notably, SYNC is pioneering in providing exact initial bacterial concentrations, achieve to 104 bacteria/ml, a capability unmatched by existing real-time techniques measuring electrochemical variations. Along with its robust performance, this earmarks SYNC as a powerful tool for applications such as antibiotic susceptibility testing, food quality monitoring, and real-time water bacteria monitoring, paving the way for enhanced microbial process management and infection control.

Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

Label-free single-cell analysis in microdroplets using a light-scattering-based optofluidic chip.

Droplet-based single-cell analysis is a very powerful tool for studying phenotypic and genomic heterogeneity at single-cell resolution for a variety of biological problems. In conventional two-phase droplet microfluidics, due to the mismatch in optical properties between oil and aqueous phases, light scattering mainly happens at the oil/water interface that disables light-scattering-based cell analysis confined in microdroplets. Detection and analysis of cells in microdroplets thus mostly rely on the fluorescence labeling of cell samples, which may suffer from complex operation, cytotoxicity, and low fluorescence stability. In this work, we propose a novel light-scattering-based droplet screening (LSDS) that can effectively detect and characterize single cells confined in droplets by adjusting the optical properties of droplets in a multiangle optofluidic chip. Theoretical and simulated calculations suggest that refractive index (RI) matching in droplet two-phase materials can reduce or eliminate droplets' scattered signals (background signal), enabling the differentiation of scattered signals from single cells and particles within droplets. Furthermore, by using a set of multiangle (from -145° to 140°) optical fibers integrated into the optofluidic chip, the scattered light properties of droplets with the RI ranging from 1.334 to 1.429 are measured. We find that the smaller the RI and size of microparticles inside droplets are, the smaller the RI difference between two-phase materials Δn is required. Especially, when Δn is smaller than 0.02, single cells in droplets can be detected and analyzed solely based on light scattering. This capability allows to accurately detect droplets containing one single cell and one single gel bead, a typical droplet encapsulation for single-cell sequencing. Altogether, this work provides a powerful platform for high-throughput label-free single-cell analysis in microdroplets for diverse single-cell related biological assays.

Genome-Wide Identification of bZIP Transcription Factors in Cymbidium ensifolium and Analysis of Their Expression under Low-Temperature Stress.

The basic leucine zipper (bZIP) transcription factors constitute the most widely distributed and conserved eukaryotic family. They play crucial roles in plant growth, development, and responses to both biotic and abiotic stresses, exerting strong regulatory control over the expression of downstream genes. In this study, a genome-wide characterization of the CebZIP transcription factor family was conducted using bioinformatic analysis. Various aspects, including physicochemical properties, phylogenetics, conserved structural domains, gene structures, chromosomal distribution, gene covariance relationships, promoter cis-acting elements, and gene expression patterns, were thoroughly analyzed. A total of 70 CebZIP genes were identified from the C. ensifolium genome, and they were randomly distributed across 18 chromosomes. The phylogenetic tree clustered them into 11 subfamilies, each exhibiting complex gene structures and conserved motifs arranged in a specific order. Nineteen pairs of duplicated genes were identified among the 70 CebZIP genes, with sixteen pairs affected by purifying selection. Cis-acting elements analysis revealed a plethora of regulatory elements associated with stress response, plant hormones, and plant growth and development. Transcriptome and qRT-PCR results demonstrated that the expression of CebZIP genes was universally up-regulated under low temperature conditions. However, the expression patterns varied among different members. This study provides theoretical references for identifying key bZIP genes in C. ensifolium that confer resistance to low-temperature stress, and lays the groundwork for further research into their broader biological functions.

Rapid and Accurate Antimicrobial Susceptibility Testing Using Label-Free Electrical Impedance-Based Microfluidic Platform.

Antimicrobial resistance has become a serious threat to the global public health. Accurate and rapid antimicrobial susceptibility testing (AST) allows evidence-based prescribing of antibiotics to improve patient care and clinical outcomes. Current culture-based AST assays are inherently limited by the doubling time of bacterial reproduction, which require at least 24 h to have a decisive result. Herein, a label-free electrical impedance-based microfluidic platform designed to expedite and streamline AST procedure for clinical practice is presented. Following a 30-min exposure of bacterial samples to antibiotics, the presented high-throughput, single-bacterium level impedance characterization platform enables a rapid 2-min AST assay. The platform facilitates accurate analysis of individual bacterial viability, as indicated by changes in electrical characteristics, thereby enabling the determination of antimicrobial resistance. Moreover, the potential clinical applicability of this platform is demonstrated by testing different E. coli strains against five antibiotics, yielding 100% categorical agreements compared to standard culture methods.

Label-free multidimensional bacterial characterization with an ultrawide detectable concentration range by microfluidic impedance cytometry.

Rapid and accurate identification of bacteria is of great importance to public health in various fields, including medical diagnostics, food safety, and environmental monitoring. However, most existing bacterial detection methods have very narrow detectable concentration ranges and limited detection information, which easily leads to wrong diagnosis and treatment. This work presents a novel high-throughput microfluidic electrical impedance-based multidimensional single-bacterium profiling system for ultrawide concentration range detection and accurate differentiation of viability and Gram types of bacteria. The electrical impedance-based microfluidic cytometry is capable of multi-frequency impedance quantification, which allows profiling of the bacteria size, concentration, and membrane impedance as an indicator of bacterial viability and Gram properties in a single flow-through interrogation. It has been demonstrated that this novel impedance cytometry has an ultrawide bacterial counting range (102-108 cells per mL), and exhibits a rapid and accurate discrimination of viability and Gram types of bacteria in a label-free manner. Escherichia coli (E. coli) has been used as an analog species for the accuracy assessment of the electrical impedance-based bacterial detection system in an authentic complex beverage matrix within 24 hours. The impedance-based quantifications of viable bacteria are consistent with those obtained by the classical bacterial colony counting method (R2 = 0.996). This work could pave the way for providing a novel microfluidic cytometry system for rapid and multidimensional bacterial detection in diverse areas.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

DUPLETS: Deformability-Assisted Dual-Particle Encapsulation Via Electrically Activated Sorting.

Co-encapsulation of bead carriers and biological cells in microfluidics has become a powerful technique for various biological assays in single-cell genomics and drug screening because of its distinct capability of single-cell confinement. However, current co-encapsulation approaches exist a trade-off between cell/bead pairing rate and probability of multiple cells in individual droplets, significantly limiting the effective throughput of single-paired cell-bead droplets production. Deformability-assisted dUal-Particle encapsuLation via Electrically acTivated Sorting (DUPLETS) system is reported to overcome this problem. The DUPLETS can differentiate the encapsulated content in individual droplets and sort out targeted droplets via a combined screening of mechanical and electrical characteristics of single droplets in label-free manners and with the highest effective throughput in comparison to current commercial platforms. The DUPLETS has been demonstrated to enrich single-paired cell-bead droplets to over 80% (above eightfold higher than current co-encapsulation techniques). It eliminates multicell droplets to 0.1% whereas up to ≈24% in 10× Chromium. It is believed that merging DUPLETS into the current co-encapsulation platforms can meaningfully elevate sample quality in terms of high purity of single-paired cell-bead droplets, low fraction of multicell droplets, and high cell viability, which can benefit a multitude of biological assay applications.

Uncovering early transcriptional regulation during adventitious root formation in Medicago sativa.

BACKGROUND: Alfalfa (Medicago sativa L.) as an important legume plant can quickly produce adventitious roots (ARs) to form new plants by cutting. But the regulatory mechanism of AR formation in alfalfa remains unclear. RESULTS: To better understand the rooting process of alfalfa cuttings, plant materials from four stages, including initial separation stage (C stage), induction stage (Y stage), AR primordium formation stage (P stage) and AR maturation stage (S stage) were collected and used for RNA-Seq. Meanwhile, three candidate genes (SAUR, VAN3 and EGLC) were selected to explore their roles in AR formation. The numbers of differentially expressed genes (DEGs) of Y-vs-C (9,724) and P-vs-Y groups (6,836) were larger than that of S-vs-P group (150), indicating highly active in the early AR formation during the complicated development process. Pathways related to cell wall and sugar metabolism, root development, cell cycle, stem cell, and protease were identified, indicating that these genes were involved in AR production. A large number of hormone-related genes associated with the formation of alfalfa ARs have also been identified, in which auxin, ABA and brassinosteroids are thought to play key regulatory roles. Comparing with TF database, it was found that AP2/ERF-ERF, bHLH, WRKY, NAC, MYB, C2H2, bZIP, GRAS played a major regulatory role in the production of ARs of alfalfa. Furthermore, three identified genes showed significant promotion effect on AR formation. CONCLUSIONS: Stimulation of stem basal cells in alfalfa by cutting induced AR production through the regulation of various hormones, transcription factors and kinases. This study provides new insights of AR formation in alfalfa and enriches gene resources in crop planting and cultivation.

Selectable encapsulated cell quantity in droplets via label-free electrical screening and impedance-activated sorting.

Single-cell encapsulation in droplets has become a powerful tool in immunotherapy, medicine discovery, and single-cell analysis, thanks to its capability for cell confinement in picoliter volumes. However, the purity and throughput of single-cell droplets are limited by random encapsulation process, which resuts in a majority of empty and multi-cells droplets. Herein we introduce the first label-free selectable cell quantity encapsulation in droplets sorting system to overcome this problem. The system utilizes a simple and reliable electrical impedance based screening (98.9% of accuracy) integrated with biocompatible acoustic sorting to select single-cell droplets, achieving 90.3% of efficiency and up to 200 â€‹Hz of throughput, by removing multi-cells (∼60% of rejection) and empty droplets (∼90% of rejection). We demonstrate the use of the droplet sorting to improve the throughput of single-cell encapsulation by ∼9-fold compared to the conventional random encapsulation process.

Genome-Wide Identification and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cymbidium ensifolium.

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the Cymbidium ensifolium genome and divided into 18 subfamilies. Most CebHLHs contain numerous cis-acting elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the CebHLHs, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 CebHLHs were differentially expressed in four different color sepals, especially CebHLH13 and CebHLH75 of the S7 subfamily. The expression profiles of CebHLH13 and CebHLH75 in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of CebHLHs in flower color formation.

Machine learning empowered multi-stress level electromechanical phenotyping for high-dimensional single cell analysis.

Microfluidics provides a powerful platform for biological analysis by harnessing the ability to precisely manipulate fluids and microparticles with integrated microsensors. Here, we introduce an imaging and impedance cell analyzer (IM2Cell), which implements single cell level impedance analysis and hydrodynamic mechanical phenotyping simultaneously. For the first time, IM2Cell demonstrates the capability of multi-stress level mechanical phenotyping. Specifically, IM2Cell is capable of characterizing cell diameter, three deformability responses, and four electrical properties. It presents high-dimensional information to give insight into subcellular components such as cell membrane, cytoplasm, cytoskeleton, and nucleus. In this work, we first validate imaging and impedance-based cell analyses separately. Then, the two techniques are combined to obtain both imaging and impedance data analyzed by machine learning method, exhibiting an improved prediction accuracy from 83.1% to 95.4% between fixed and living MDA-MB-231 breast cancer cells. Next, IM2Cell demonstrates 91.2% classification accuracy in a mixture of unlabeled MCF-10A, MCF-7, and MDA-MB-231 cell lines. Finally, an application demonstrates the potential of IM2Cell for the deformability studies of peripheral blood mononuclear cells (PBMCs) subpopulations without cumbersome isolation or labeling steps.

Tunable and Dynamic Optofluidic Microlens Arrays Based on Droplets.

Microlens arrays (MLAs) are acquiring a key role in the micro-optical system, which have been widely applied in the fields of imaging processing, light extraction, biochemical sensing, and display technology. Compared with solid MLAs, liquid MLAs have received extensive attention due to their natural smooth interface and adjustability. However, manufacturing tunable liquid MLAs with ideal structures is still a key challenge for current technologies. In this paper, a novel and simple optofluidic method is demonstrated, enabling the tunable focusing and high-quality imaging of liquid MLAs. Tunable droplets are fabricated and self-assembled into arrays as the MLAs, which can be easily adjusted to focus, form images, and display different focal lengths. Tuning of MLAs' focusing properties (range from 550 to 5370 µm) is demonstrated by changing the refractive index (RI) of the droplets with a fixed size of 200 µm, which can be changed by adjusting the flow rates of the two branch streams. Also, the corresponding numerical apertures of the MLAs range from 0.026 to 0.26. Furthermore, the MLAs' functionality for microparticle imaging applications is also illustrated. Combining the MLAs with a 4× objective, microparticle imaging is magnified two times, and the resolution has also been improved on the original basis. Besides, both the size and RI of the MLAs in an optofluidic chip can be further adjusted to detect samples at different positions. These MLAs have the merits of high optical performance, a simple fabrication procedure, easy integration, and good tunability. Thus, it shows promising opportunities for many applications, such as adaptive imaging and sensing.

A Systematic Study of Size Correlation and Young's Modulus Sensitivity for Cellular Mechanical Phenotyping by Microfluidic Approaches.

Cellular mechanical properties are a class of intrinsic biophysical markers for cell state and health. Microfluidic mechanical phenotyping methods have emerged as promising tools to overcome the challenges of low throughput and high demand for manual skills in conventional approaches. In this work, two types of microfluidic cellular mechanical phenotyping methods, contactless hydro-stretching deformability cytometry (lh-DC) and contact constriction deformability cytometry (cc-DC) are comprehensively studied and compared. Polymerized hydrogel beads with defined sizes are used to characterize a strong negative correlation between size and deformability in cc-DC (r = -0.95), while lh-DC presents a weak positive correlation (r = 0.13). Young's modulus sensitivity in cc-DC is size-dependent while it is a constant in lh-DC. Moreover, the deformability assessment for human breast cell line mixture suggests the lh-DC exhibits better differentiation capability of cells with different size distributions, while cc-DC provides higher sensitivity to identify cellular mechanical changes within a single cell line. This work is the first to present a quantitative study and comparison of size correlation and Young's modulus sensitivity of contactless and contact microfluidic mechanical phenotyping methods, which provides guidance to choose the most suitable cellular mechanical phenotyping platform for specific cell analysis applications.

Automatic Microfluidic Cell Wash Platform for Purifying Cells in Suspension: Puriogen.

Cell wash is an essential cell sample preparation procedure to eliminate or minimize interfering substances for various subsequent cell analyses. The commonly used cell wash method is centrifugation which separates cells from other biomolecules in a solution with manual pipetting and has many drawbacks such as being labor-intensive and time-consuming with substantial cell loss and cell clumping. To overcome these issues, a centrifuge-free and automatic cell wash platform for cell purity generation, termed Puriogen, has been developed in this work. Compared with other developed products such as AcouWash, Puriogen can process samples with a high throughput of above 500 µL/min. Puriogen utilizes a uniquely designed inertial microfluidic device to complete the automatic cell wash procedure. One single-cell wash procedure with the Puriogen platform can remove more than 90% ambient proteins and nucleic acids from the cell sample. It can also remove most of the residual fluorescent dye after cell staining to significantly reduce the background signals for subsequent cell analysis. By removing the dead cell debris, it can increase the live cell percentage in the sample by 2-fold. Moreover, the percentage of single-cell population is also increased by 20% because of further disassociation of small-cell aggregates (e.g., doublets and triplets) into singlets. To freely adjust cell concentrations, the Puriogen platform can concentrate cells 5 times in a single flow-through process. The presented Puriogen cell wash solution has broad applications in cell preparation with its excellent simplicity in operation and wash efficiency, especially in single-cell sequencing.

Genomes of leafy and leafless Platanthera orchids illuminate the evolution of mycoheterotrophy.

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.

The Melastoma dodecandrum genome and the evolution of Myrtales.

Melastomataceae has abundant morphological diversity with high economic and ornamental merit in Myrtales. The phylogenetic position of Myrtales is still contested. Here, we report the chromosome-level genome assembly of Melastoma dodecandrum in Melastomataceae. The assembled genome size is 299.81 Mb with a contig N50 value of 3.00 Mb. Genome evolution analysis indicated that M. dodecandrum, Eucalyptus grandis, and Punica granatum were clustered into a clade of Myrtales and formed a sister group with the ancestor of fabids and malvids. We found that M. dodecandrum experienced four whole-genome polyploidization events: the ancient event was shared with most eudicots, one event was shared with Myrtales, and the other two events were unique to M. dodecandrum. Moreover, we identified MADS-box genes and found that the AP1-like genes expanded, and AP3-like genes might have undergone subfunctionalization. The SUAR63-like genes and AG-like genes showed different expression patterns in stamens, which may be associated with heteranthery. In addition, we found that LAZY1-like genes were involved in the negative regulation of stem branching development, which may be related to its creeping features. Our study sheds new light on the evolution of Melastomataceae and Myrtales, which provides a comprehensive genetic resource for future research.

Genome-Wide Identification and Expression Analysis of Terpene Synthase Genes in Cymbidium faberi.

Terpene synthases (TPSs) are essential for forming terpenes, which play numerous functional roles in attracting pollinators, defending plants, and moderating the interaction between plants. TPSs have been reported in some orchids, but genome-wide identification of terpenes in Cymbidium faberi is still lacking. In this study, 32 putative TPS genes were classified in C. faberi and divided into three subfamilies (TPS-a, TPS-b, and TPS-e/f). Motif and gene structure analysis revealed that most CfTPS genes had the conserved aspartate-rich DDxxD motif. TPS genes in the TPS-a and TPS-b subfamilies had variations in the RRX8W motif. Most cis-elements of CfTPS genes were found in the phytohormone responsiveness category, and MYC contained most of the numbers associated with MeJA responsiveness. The Ka/Ks ratios of 12/13 CfTPS gene pairs were less than one, indicated that most CfTPS genes have undergone negative selection. The tissue-specific expression patterns showed that 28 genes were expressed in at least one tissue in C. faberi, and TPS genes were most highly expressed in flowers, followed by leaves and pseudobulbs. In addition, four CfTPS genes were selected for the real-time reverse transcription quantitative PCR (RT-qPCR) experiment. The results revealed that CfTPS12, CfTPS18, CfTPS23, and CfTPS28 were mainly expressed in the full flowering stage. CfTPS18 could convert GPP to ß-myrcene, geraniol, and α-pinene in vitro. These findings of CfTPS genes of C. faberi may provide valuable information for further studies on TPSs in orchids.

Genome-Wide Identification of the MYB Gene Family in Cymbidiumensifolium and Its Expression Analysis in Different Flower Colors.

MYB transcription factors of plants play important roles in flavonoid synthesis, aroma regulation, floral organ morphogenesis, and responses to biotic and abiotic stresses. Cymbidium ensifolium is a perennial herbaceous plant belonging to Orchidaceae, with special flower colors and high ornamental value. In this study, a total of 136 CeMYB transcription factors were identified from the genome of C. ensifolium, including 27 1R-MYBs, 102 R2R3-MYBs, 2 3R-MYBs, 2 4R-MYBs, and 3 atypical MYBs. Through phylogenetic analysis in combination with MYB in Arabidopsis thaliana, 20 clusters were obtained, indicating that these CeMYBs may have a variety of biological functions. The 136 CeMYBs were distributed on 18 chromosomes, and the conserved domain analysis showed that they harbored typical amino acid sequence repeats. The motif prediction revealed that multiple conserved elements were mostly located in the N-terminal of CeMYBs, suggesting their functions to be relatively conserved. CeMYBs harbored introns ranging from 0 to 13 and contained a large number of stress- and hormone-responsive cis-acting elements in the promoter regions. The subcellular localization prediction demonstrated that most of CeMYBs were positioned in the nucleus. The analysis of the CeMYBs expression based on transcriptome data showed that CeMYB52, and CeMYB104 of the S6 subfamily may be the key genes leading to flower color variation. The results lay a foundation for the study of MYB transcription factors of C. ensifolium and provide valuable information for further investigations of the potential function of MYB genes in the process of anthocyanin biosynthesis.