In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells.

Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.

Single-Chain Conjugated Polymer Guests Confined inside Metal-Organic Frameworks (MOFs): Boosting the Detection and Degradation of a Sulfur Mustard Simulant.

Real-time detection and effective degradation of toxic gases have attracted considerable attention in environmental monitoring and human health. Here, we demonstrate a solvent-assisted dynamic assembly strategy to strongly enhance the detection and degradation performance for 2-chloroethyl ethyl sulfide (CEES, as a sulfur mustard simulant) via confinement of a conjugated polymer in metal-organic frameworks (MOFs). The conjugated polymer poly(9,9-di-n-octylfluorene-altbenzothiadiazole) (F8BT) is infiltrated into one-dimensional nanochannels of the Zr-based topological MOF NU-1000 in a single-chain manner, which is caused by the nanoconfinement effect and the steric hindrance between 9,9-dioctylfluorene units and benzothiadiazole units. The obtained F8BT⊂NU-1000 composites provide a high specific surface area and abundant active sites. Based on the cooperative effect of F8BT and NU-1000, rapid and sensitive detection of CEES has been achieved. Moreover, the F8BT⊂NU-1000 composites can selectively oxidize CEES into 2-chloroethyl ethyl sulfoxide (CEESO) under mild photooxidation conditions. Overall, this study opens a new avenue for the fabrication of conjugated polymer/MOF hybrid materials that show great potential for the sensitive detection and effective removal of hazardous chemicals.

Ultrasensitive Point-of-Care Detection of Protein Markers Using an Aptamer-CRISPR/Cas12a-Regulated Liquid Crystal Sensor (ALICS).

Despite extensive efforts, point-of-care testing (POCT) of protein markers with high sensitivity and specificity and at a low cost remains challenging. In this work, we developed an aptamer-CRISPR/Cas12a-regulated liquid crystal sensor (ALICS), which achieved ultrasensitive protein detection using a smartphone-coupled portable device. Specifically, a DNA probe that contained an aptamer sequence for the protein target and an activation sequence for the Cas12a-crRNA complex was prefixed on a substrate and was released in the presence of target. The activation sequence of the DNA probe then bound to the Cas12a-crRNA complex to activate the collateral cleavage reaction, producing a bright-to-dark optical change in a DNA-functionalized liquid crystal interface. The optical image was captured by a smartphone for quantification of the target concentration. For the two model proteins, SARS-CoV-2 nucleocapsid protein (N protein) and carcino-embryonic antigen (CEA), ALICS achieved detection limits of 0.4 and 20 pg/mL, respectively, which are higher than the typical sensitivity of the SARS-CoV-2 test and the clinical CEA test. In the clinical sample tests, ALICS also exhibited superior performances compared to those of the commercial ELISA and lateral flow test kits. Overall, ALICS represents an ultrasensitive and cost-effective platform for POCT, showing a great potential for pathogen detection and disease monitoring under resource-limited conditions.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Monodispersed and Monofunctionalized DNA-Caged Au Nano-Clusters with Enhanced Optical Properties for STED Imaging.

The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40 nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugates only achieved ≈55 nm resolution and yielded spotted, poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.

Dual anti-angiogenic and anti-inflammatory action of tRNA-Cys-5-0007 in ocular vascular disease.

BACKGROUND: Intravitreal injections of angiogenesis inhibitors have proved efficacious in the majority of patients with ocular angiogenesis. However, one-fourth of all treated patients fail to derive benefits from intravitreal injections. tRNA-derived small RNA (tsRNA) emerges as a crucial class of non-coding RNA molecules, orchestrating key roles in the progression of human diseases by modulating multiple targets. Through our prior sequencing analyses and bioinformatics predictions, tRNA-Cys-5-0007 has shown as a potential regulator of ocular angiogenesis. This study endeavors to elucidate the precise role of tRNA-Cys-5-0007 in the context of ocular angiogenesis. METHODS: Quantitative reverse transcription PCR (qRT-PCR) assays were employed to detect tRNA-Cys-5-0007expression. EdU assays, sprouting assays, transwell assays, and Matrigel assays were conducted to elucidate the involvement of tRNA-Cys-5-0007 in endothelial angiogenic effects. STZ-induced diabetic model, OIR model, and laser-induced CNV model were utilized to replicate the pivotal features of ocular vascular diseases and evaluate the influence of tRNA-Cys-5-0007 on ocular angiogenesis and inflammatory responses. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were employed to elucidate the anti-angiogenic mechanism of tRNA-Cys-5-0007. Exosomal formulation was employed to enhance the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. RESULTS: tRNA-Cys-5-0007 expression was down-regulated under angiogenic conditions. Conversely, tRNA-Cys-5-0007 overexpression exhibited anti-angiogenic effects in retinal endothelial cells, as evidenced by reduced proliferation, sprouting, migration, and tube formation abilities. In diabetic, laser-induced CNV, and OIR models, tRNA-Cys-5-0007 overexpression led to decreased ocular vessel leakage, inhibited angiogenesis, and reduced ocular inflammation. Mechanistically, these effects were attributed to the targeting of vascular endothelial growth factor A (VEGFA) and TGF-ß1 by tRNA-Cys-5-0007. The utilization of an exosomal formulation further potentiated the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. CONCLUSIONS: Concurrent targeting of tRNA-Cys-5-0007 for anti-angiogenic and anti-inflammatory therapy holds promise for enhancing the effectiveness of current anti-angiogenic therapy.

Solar-Driven Multistage Device Integrating Dropwise Condensation and Guided Water Transport for Efficient Freshwater and Salt Collection.

Interfacial solar vapor generation (ISVG) is an emerging technology to alleviate the global freshwater crisis. However, high-cost, low freshwater collection rate, and salt-blockage issues significantly hinder the practical application of solar-driven desalination devices based on ISVG. Herein, with a low-cost copper plate (CP), nonwoven fabric (NWF), and insulating ethylene-vinyl acetate foam (EVA foam), a multistage device is elaborately fabricated for highly efficient simultaneous freshwater and salt collection. In the designed solar-driven device, a superhydrophobic copper plate (SH-CP) serves as the condensation layer, facilitating rapid mass and heat transfer through dropwise condensation. Moreover, the hydrophilic NWF is designed with rational hydrophobic zones and specific high-salinity solution outlets (Design-NWF) to act as the water evaporation layer and facilitate directional salt collection. As a result, the multistage evaporator with eight stages exhibits a high water collection rate of 2.25 kg m-2 h-1 under 1 sun irradiation. In addition, the desalination device based on the eight-stage evaporator obtains a water collection rate of 13.44 kg m-2 and a salt collection rate of 1.77 kg m-2 per day under natural irradiation. More importantly, it can maintain a steady production for 15 days without obvious performance decay. This bifunctional multistage device provides a feasible and efficient approach for simultaneous desalination and solute collection.

ß-arrestin-dependent PI(4,5)P2 synthesis boosts GPCR endocytosis.

ß-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that ß-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that ß-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of ß-arrestin revealed that endogenous ß-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/ß-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, ß-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

Hyperbranched Polymer Dots with Strong Absorption and High Fluorescence Quantum Yield for In Vivo NIR-II Imaging.

In order to improve the fluorescence quantum yield (QY) of NIR-II-emitting nanoparticles, D-A-D fluorophores are typically linked to intramolecular rotatable units to reduce aggregation-induced quenching. However, incorporating such units often leads to a twisted molecular backbone, which affects the coupling within the D-A-D unit and, as a result, lowers the absorption. Here, we overcome this limitation by cross-linking the NIR-II fluorophores to form a 2D polymer network, which simultaneously achieves a high QY by well-controlled fluorophore separation and strong absorption by restricting intramolecular distortion. Using the strategy, we developed polymer dots with the highest NIR-II single-particle brightness among reported D-A-D-based nanoparticles and applied them for imaging of hindlimb vasculatures and tumors as well as fluorescence-guided tumor resection. The high brightness of the polymer dots offered exceptional image quality and excellent surgical results, showing a promising performance for these applications.

Novel Ultralow-Potential Electrochemiluminescence Aptasensor for the Highly Sensitive Detection of Zearalenone Using a Resonance Energy Transfer System.

An ultralow-potential electrochemiluminescence (ECL) aptasensor has been designed for zearalenone (ZEN) assay based on a resonance energy transfer (RET) system with SnS2 QDs/g-C3N4 as a novel luminophore and CuO/NH2-UiO-66 as a dual-quencher. SnS2 QDs were loaded onto g-C3N4 nanosheets and enhanced the ECL luminescence via strong synergistic effects under an ultralow potential. The UV-vis absorption spectrum of CuO/NH2-UiO-66 exhibits considerable overlap with the ECL emission spectrum of SnS2 QDs/g-C3N4, an important consideration for the RET process. In order to stimulate RET, the ZEN aptamer and complementary DNA are introduced for conjugation between the donor and the acceptor. With the binding interaction between ZEN by its aptamer, CuO/NH2-UiO-66 is removed from the electrode surface, resulting in the inhibition of the RET system and an increase in the ECL signal. Under optimal conditions, the as-prepared aptasensor quantified ZEN from 0.5 µg·mL-1 to 0.1 fg·mL-1 with a low limit of detection of 0.085 fg·mL-1, and it exhibited good stability, excellent specificity, high reproducibility, and desirable practicality. The sensing strategy provides a method for mycotoxins assay to monitor food safety.

Genome-Wide Identification, Evolution, and Expression Analysis of the WD40 Subfamily in Oryza Genus.

The WD40 superfamily is widely found in eukaryotes and has essential subunits that serve as scaffolds for protein complexes. WD40 proteins play important regulatory roles in plant development and physiological processes, such as transcription regulation and signal transduction; it is also involved in anthocyanin biosynthesis. In rice, only OsTTG1 was found to be associated with anthocyanin biosynthesis, and evolutionary analysis of the WD40 gene family in multiple species is less studied. Here, a genome-wide analysis of the subfamily belonging to WD40-TTG1 was performed in nine AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, Oryza glumaepatula, Oryza nivara, and Oryza longistaminata. In this study, 383 WD40 genes in the Oryza genus were identified, and they were classified into four groups by phylogenetic analysis, with most members in group C and group D. They were found to be unevenly distributed across 12 chromosomes. A total of 39 collinear gene pairs were identified in the Oryza genus, and all were segmental duplications. WD40s had similar expansion patterns in the Oryza genus. Ka/Ks analyses indicated that they had undergone mainly purifying selection during evolution. Furthermore, WD40s in the Oryza genus have similar evolutionary patterns, so Oryza sativa ssp. indica was used as a model species for further analysis. The cis-acting elements analysis showed that many genes were related to jasmonic acid and light response. Among them, OsiWD40-26/37/42 contained elements of flavonoid synthesis, and OsiWD40-15 had MYB binding sites, indicating that they might be related to anthocyanin synthesis. The expression profile analysis at different stages revealed that most OsiWD40s were expressed in leaves, roots, and panicles. The expression of OsiWD40s was further analyzed by qRT-PCR in 9311 (indica) under various hormone treatments and abiotic stresses. OsiWD40-24 was found to be responsive to both phytohormones and abiotic stresses, suggesting that it might play an important role in plant stress resistance. And many OsiWD40s might be more involved in cold stress tolerance. These findings contribute to a better understanding of the evolution of the WD40 subfamily. The analyzed candidate genes can be used for the exploration of practical applications in rice, such as cultivar culture for colored rice, stress tolerance varieties, and morphological marker development.

Genome-Wide Identification and Evolution Analysis of the CYP76 Subfamily in Rice (Oryza sativa).

The CYP76 subfamily, a member of the CYP superfamily, plays crucial roles in the biosynthesis of phytohormones in plants, involving biosynthesis of secondary metabolites, hormone signaling, and response to environmental stresses. Here, we conducted a genome-wide analysis of the CYP76 subfamily in seven AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, and Oryza glumaepatula. These were identified and classified into three groups, and it was found that Group 1 contained the largest number of members. Analysis of cis-acting elements revealed a large number of elements related to jasmonic acid and light response. The gene duplication analysis revealed that the CYP76 subfamily expanded mainly in SD/WGD and tandem forms and underwent strong purifying selection during evolution. Expression pattern analysis of OsCYP76s in various developmental stages revealed that the majority of OsCYP76s exhibit relatively restricted expression patterns in leaves and roots. We further analyzed the expression of CYP76s in O. sativa, japonica, and O. sativa, indica under cold, flooding, drought, and salt abiotic stresses by qRT-PCR. We found that OsCYP76-11 showed a huge increase in relative expression after drought and salt stresses. After flooding stress, OsiCYP76-4 showed a greater increase in expression compared to other genes. CYP76 in japonica and indica showed different response patterns to the same abiotic stresses, revealing functional divergence in the gene family during evolution; these may be the key genes responsible for the differences in tolerance to indica japonica. Our results provide valuable insights into the functional diversity and evolutionary history of the CYP76 subfamily and pave the way for the development of new strategies for improving stress tolerance and agronomic traits in rice.

High-Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super-Resolution Imaging.

The spatial resolution of single-molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single-particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4-9 frame binning, we achieved a 2-3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.

Isoelectric Electroencephalography in Infants and Toddlers during Anesthesia for Surgery: An International Observational Study.

BACKGROUND: Intraoperative isoelectric electroencephalography (EEG) has been associated with hypotension and postoperative delirium in adults. This international prospective observational study sought to determine the prevalence of isoelectric EEG in young children during anesthesia. The authors hypothesized that the prevalence of isoelectric events would be common worldwide and associated with certain anesthetic practices and intraoperative hypotension. METHODS: Fifteen hospitals enrolled patients age 36 months or younger for surgery using sevoflurane or propofol anesthetic. Frontal four-channel EEG was recorded for isoelectric events. Demographics, anesthetic, emergence behavior, and Pediatric Quality of Life variables were analyzed for association with isoelectric events. RESULTS: Isoelectric events occurred in 32% (206 of 648) of patients, varied significantly among sites (9 to 88%), and were most prevalent during pre-incision (117 of 628; 19%) and surgical maintenance (117 of 643; 18%). Isoelectric events were more likely with infants younger than 3 months (odds ratio, 4.4; 95% CI, 2.57 to 7.4; P < 0.001), endotracheal tube use (odds ratio, 1.78; 95% CI, 1.16 to 2.73; P = 0.008), and propofol bolus for airway placement after sevoflurane induction (odds ratio, 2.92; 95% CI, 1.78 to 4.8; P < 0.001), and less likely with use of muscle relaxant for intubation (odds ratio, 0.67; 95% CI, 0.46 to 0.99; P = 0.046]. Expired sevoflurane was higher in patients with isoelectric events during preincision (mean difference, 0.2%; 95% CI, 0.1 to 0.4; P = 0.005) and surgical maintenance (mean difference, 0.2%; 95% CI, 0.1 to 0.3; P = 0.002). Isoelectric events were associated with moderate (8 of 12, 67%) and severe hypotension (11 of 18, 61%) during preincision (odds ratio, 4.6; 95% CI, 1.30 to 16.1; P = 0.018) (odds ratio, 3.54; 95% CI, 1.27 to 9.9; P = 0.015) and surgical maintenance (odds ratio, 3.64; 95% CI, 1.71 to 7.8; P = 0.001) (odds ratio, 7.1; 95% CI, 1.78 to 28.1; P = 0.005), and lower Pediatric Quality of Life scores at baseline in patients 0 to 12 months (median of differences, -3.5; 95% CI, -6.2 to -0.7; P = 0.008) and 25 to 36 months (median of differences, -6.3; 95% CI, -10.4 to -2.1; P = 0.003) and 30-day follow-up in 0 to 12 months (median of differences, -2.8; 95% CI, -4.9 to 0; P = 0.036). Isoelectric events were not associated with emergence behavior or anesthetic (sevoflurane vs. propofol). CONCLUSIONS: Isoelectric events were common worldwide in young children during anesthesia and associated with age, specific anesthetic practices, and intraoperative hypotension.

A Cloud-Computing-Based Portable Networked Ground Station System for Microsatellites.

Microsatellites have attracted a large number of scholars and engineers because of their portability and distribution characteristics. The ground station suitable for microsatellite service has become an important research topic. In this paper, we propose a networked ground station and verify it on our own microsatellite. The specific networked ground station system consists of multiple ground nodes. They can work together to complete data transmission tasks with higher efficiency. After describing our microsatellite project, a reasonable distribution of ground nodes is given. A cloud computing model is used to realize the coordination of multiple ground nodes. An adaptive communication system between satellites and ground stations is used to increase link efficiency. Extensive on-orbit experiments were used to validate our design. The experimental results show that our networked ground station has excellent performance in data transmission capability. Finally, the specific cloud-computing-based ground station network successfully completes our satellite mission.

Multimode Time-Resolved Superresolution Microscopy Revealing Chain Packing and Anisotropic Single Carrier Transport in Conjugated Polymer Nanowires.

Here, we developed a novel, multimode superresolution method to perform full-scale structural mapping and measure the energy landscape for single carrier transport along conjugated polymer nanowires. Through quenching of the local emission, the motion of a single photogenerated hole was tracked using blinking-assisted localization microscopy. Then, utilizing binding and unbinding dynamics of quenchers onto the nanowires, local emission spectra were collected sequentially and assembled to create a superresolution map of emission sites throughout the structure. The hole polaron trajectories were overlaid with the superresolution maps to correlate structures with charge transport properties. Using this method, we compared the efficiency of inter- and intrachain hole transport inside the nanowires and for the first time directly measured the depth of carrier traps originated from torsional disorder and chemical defects.

Genome-Wide Identification of the AGC Protein Kinase Gene Family Related to Photosynthesis in Rice (Oryza sativa).

The cAMP-dependent protein kinase A, cGMP-dependent protein kinase G and phospholipid-dependent protein kinase C (AGC) perform various functions in plants, involving growth, immunity, apoptosis and stress response. AGC gene family is well described in Arabidopsis, however, limited information is provided about AGC genes in rice, an important cereal crop. This research studied the AGC gene family in the AA genome species: Oryza sativa ssp. japonica, Oryza sativa ssp. indica, Oryza nivara, Oryza rufipogon, Oryza glaberrima, Oryza meridionalis, Oryza barthii, Oryza glumaepatula and Oryza longistaminata were searched and classified into six subfamilies, and it was found that these species have similar numbers of members. The analysis of gene duplication and selection pressure indicated that the AGC gene family expanded mainly by segmental or whole genome duplication (WGD), with purifying selection during the long evolutionary period. RNA-seq analysis revealed that OsAGCs of subfamily V were specifically highly expressed in leaves, and the expression patterns of these genes were compared with that of photosynthesis-related genes using qRT-PCR, discovered that OsAGC9, OsAGC20, and OsAGC22 might participate in photosynthesis. These results provide an informative perspective for exploring the evolutionary of AGC gene family and its practical application in rice.

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer.

Extracellular vesicles (EVs) are membranous particles released by most cells in our body, which are involved in many cell-to-cell signaling processes. Given the nanometer sizes and heterogeneity of EVs, highly sensitive methods with single-molecule resolution are fundamental to investigating their biophysical properties. Here, we demonstrate the sizing of EVs using a fluorescence-based flow analyzer with single-molecule sensitivity. Using a dye that selectively partitions into the vesicle's membrane, we show that the fluorescence intensity of a vesicle is proportional to its diameter. We discuss the constraints in sample preparation which are inherent to sizing nanoscale vesicles with a fluorescent membrane dye and propose several guidelines to improve data consistency. After optimizing staining conditions, we were able to measure the size of vesicles in the range ∼35-300 nm, covering the spectrum of EV sizes. Lastly, we developed a method to correct the signal intensity from each vesicle based on its traveling speed inside the microfluidic channel, by operating at a high sampling rate (10 kHz) and measuring the time required for the particle to cross the laser beam. Using this correction, we obtained a threefold greater accuracy in EV sizing, with a precision of ±15-25%.

Improving the Accuracy of Pdot-Based Continuous Glucose Monitoring by Using External Ratiometric Calibration.

Continuous glucose monitoring (CGM) allows type I and II diabetes patients to track changes in their glucose levels, allowing detection of impending hypoglycemia or hyperglycemia. Polymer dots (Pdots) are candidates for use in implanted CGM systems due to their exceptional brightness, photostability, sensitivity, and biocompatibility. However, Pdot glucose transducers are oxygen-dependent, and changes in tissue oxygen levels affect their measurement accuracy. Here, we describe an external ratiometric calibration method that corrects for changes in tissue oxygen levels to improve measurement accuracy. This method uses the ratio of oxygen concentrations inside and outside the Pdot glucose transducer as an indicator of glucose concentration to correct for signal deviations caused by tissue oxygen fluctuations. A second oxygen-sensitive Pdot that is not conjugated with glucose oxidase is used to measure the oxygen concentration outside the Pdot glucose transducer. We describe the theoretical basis for this approach and demonstrate its effectiveness experimentally in a subcutaneous mouse implant model. This external ratiometric system achieves higher accuracy glucose measurements than previous Pdot-based CGM systems and comparable accuracy to current commercial CGM systems, demonstrating the utility of the external ratiometric calibration strategy.

Sequential Ensemble-Decision Aliquot Ranking Isolation and Fluorescence In Situ Hybridization Identification of Rare Cells from Blood by Using Concentrated Peripheral Blood Mononuclear Cells.

Isolation and analysis of circulating rare cells is a promising approach for early detection of cancer and other diseases and for prenatal diagnosis. Isolation of rare cells is usually difficult due to their heterogeneity as well as their low abundance in peripheral blood. We previously reported a two-stage ensemble-decision aliquot ranking platform (S-eDAR) for isolating circulating tumor cells from whole blood with high throughput, high recovery rate (>90%), and good purity (>70%), allowing detection of low surface antigen-expressing cancer cells linked to metastasis. However, due to the scarcity of these cells, large sample volumes and large quantities of antibodies were required to isolate sufficient cells for downstream analysis. Here, we drastically increased the number of nucleated cells analyzed by first concentrating peripheral blood mononuclear cells (PBMCs) from whole blood by density gradient centrifugation. The S-eDAR platform was capable of isolating rare cells from concentrated PBMCs (108/mL, equivalent to processing ∼20 mL of whole blood in the 1 mL sample volume used by our instrument) at a high recovery rate (>85%). We then applied the S-eDAR platform for isolating rare fetal nucleated red blood cells (fNRBCs) from concentrated PBMCs spiked with umbilical cord blood cells and confirmed fNRBC recovery by immunostaining and fluorescence in situ hybridization, demonstrating the potential of the S-eDAR system for isolating rare fetal cells from maternal PBMCs to improve noninvasive prenatal diagnosis.