Optofluidic time-stretch imaging flow cytometry with a real-time storage rate beyond 5.9 GB/s.

Optofluidic time-stretch imaging flow cytometry (OTS-IFC) provides a suitable solution for high-precision cell analysis and high-sensitivity detection of rare cells due to its high-throughput and continuous image acquisition. However, transferring and storing continuous big data streams remains a challenge. In this study, we designed a high-speed streaming storage strategy to store OTS-IFC data in real-time, overcoming the imbalance between the fast generation speed in the data acquisition and processing subsystem and the comparatively slower storage speed in the transmission and storage subsystem. This strategy, utilizing an asynchronous buffer structure built on the producer-consumer model, optimizes memory usage for enhanced data throughput and stability. We evaluated the storage performance of the high-speed streaming storage strategy in ultra-large-scale blood cell imaging on a common commercial device. The experimental results show that it can provide a continuous data throughput of up to 5891 MB/s.

Recovery of Li and Co in Waste Lithium Cobalt Oxide-Based Battery Using H1.6Mn1.6O4.

H1.6Mn1.6O4 lithium-ion screen adsorbents were synthesized by soft chemical synthesis and solid phase calcination and then applied to the recovery of metal Li and Co from waste cathode materials of a lithium cobalt oxide-based battery. The leaching experiments of cobalt and lithium from cathode materials by a citrate hydrogen peroxide system and tartaric acid system were investigated. The experimental results showed that under the citrate hydrogen peroxide system, when the temperature was 90 °C, the rotation speed was 600 r·min-1 and the solid-liquid ratio was 10 g·1 L-1, the leaching rate of Co and Li could reach 86.21% and 96.9%, respectively. Under the tartaric acid system, the leaching rates of Co and Li were 90.34% and 92.47%, respectively, under the previous operating conditions. The adsorption results of the lithium-ion screen showed that the adsorbents were highly selective for Li+, and the maximum adsorption capacities were 38.05 mg·g-1. In the process of lithium removal, the dissolution rate of lithium was about 91%, and the results of multiple cycles showed that the stability of the adsorbent was high. The recovery results showed that the purity of LiCl, Li2CO3 and CoCl2 crystals could reach 93%, 99.59% and 87.9%, respectively. LiCoO2 was regenerated by the sol-gel method. XRD results showed that the regenerated LiCoO2 had the advantages of higher crystallinity and less impurity.

Single-Particle Tracking Reveals the Interplay between HIV-1 Reverse Transcription and Uncoating.

Reverse transcription uses the reverse transcriptase enzyme to synthesize deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template. This plays an essential role in viral replication. There are still, however, many unknown facts regarding the timing and dynamic processes involved in this life stage. Here, three types of dual-fluorescence human immunodeficiency virus type-1 (HIV-1) particles were constructed with high infectivity, and the sequential process of reverse transcription was observed by real-time imaging of a single HIV-1 particle. Viral uncoating occurred at 60-120 min post infection. Subsequently, at 120-180 min post infection, the viral genome was separated into two parts and reverse-transcribed to generate a DNA product. Nevirapine (NVP), a reverse transcriptase inhibitor, can delay the dynamic process. This study revealed a delicate, sequential, and complex relationship between uncoating and reverse transcription, which may facilitate the development of antiviral drugs.

Synthesis of Dual-Emitting CdZnSe/Mn:ZnS Quantum Dots for Sensing the pH Change in Live Cells.

Intracellular pH is an important regulator of cell function, and its subtle changes may greatly affect cell activities and cause diseases. Reliable imaging of intracellular pH remains a huge challenge. Dual-emitting Mn2+-doped quantum dots (QDs) can be directly used as a ratiometric fluorescent probe without further modification, but they displayed low performance in terms of photoluminescence, stability, and intensity ratio regulation. Here, we report intrinsic dual-emitting CdZnSe/Mn:ZnS QDs with high photoluminescence efficiency, good stability, and biocompatibility. The emission intensity ratio was selectively regulated by Mn2+ doping. Because of aggregation-induced quenching of QDs, the exciton emission of CdZnSe/Mn:ZnS QDs (471 nm) was sensitive to pH, while the Mn2+-doped emission (606 nm) was passive to pH, which was probably due to little self-quenching in Mn2+-doped emission caused by weak Mn-Mn coupling interaction. Dual-emitting CdZnSe/Mn:ZnS QDs exhibited excellent pH-responsiveness in the range of pH 4.0 to 12.0 and were used for pH imaging in live HeLa cells. When the pH value of HeLa cells changed from 5.0 to 9.0, the emission changed from red to blue. Furthermore, these dual-emitting CdZnSe/Mn:ZnS QDs can provide a versatile platform for biosensors and biological imaging.

Construction of ratiometric Si-Mn:ZnSe nanoparticles for the immunoassay of SARS-CoV-2 spike protein.

The continuing global spread of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, has led to an unprecedented global health crisis. Effective and affordable methods are needed to diagnose SARS-CoV-2 infection. In this work, a ratiometric fluorescence probe, Si-Mn:ZnSe nanoparticles, was constructed through the electrostatic interaction between Si dots and Mn:ZnSe QDs, and the fluorescence of Mn:ZnSe QDs has a specifical response to H2O2. An immunocomplex was formed by the recognition of capture antibody/spike (S) protein/spike neutralizing antibody/biotinylated second antibody/streptavidin/biotinylated catalase (CAT). In the presence of S protein, CAT effectively catalyzed the decomposition of H2O2 in the system, and the fluorescence of Mn:ZnSe QDs was not specifically quenched. Based on this principle, a ratiometric immunoassay of SARS-CoV-2 S protein was established. The sensitivity of the proposed ELISA method was comparable to that of the commercial kit. In addition, this method can effectively distinguish the pseudo-SARS-CoV-2 virus and other pseudovirus. Therefore, this method provided a reliable and potential direction for diagnosing SARS-CoV-2 infection.

Novel Method of Clickable Quantum Dot Construction for Bioorthogonal Labeling.

Bioorthogonal chemistry has been considered as a powerful tool for biomolecule labeling due to its site specificity, moderate reaction conditions, high yield, and simple post-treatment. Covalent coupling is commonly used to modify quantum dots (QDs) with bioorthogonal functional group (azide or cycloalkyne), but it has a negative effect in the decrease of QDs' quantum yield and stability and increase of QDs' hydrodynamic diameter. To overcome these disadvantages, we propose a novel method for the preparation of two kinds of clickable QDs by the strong interaction of -SH with metal ions. One system involves azide-DNA-functionalized QDs, which are used for bioconjugation with dibenzocyclooctyne (DBCO)-modified glucose oxidase (GOx) to form a GOx-QDs complex. After bioconjugation, the stability of QDs was improved, and the activity of GOx was also enhanced. The GOx-QDs complex was used for rapid detection of blood glucose by spectroscopy, naked eye, and paper-based analytical devices. The second system involves DBCO-DNA-functionalized QDs, which are used for an in situ bioorthogonal labeling of HeLa cells through metabolic oligosaccharide engineering. Therefore, these clickable QDs based on DNA functionalization can be applied for rapid and effective labeling of biomolecules of interest.

SARS-CoV-2 spike protein receptor-binding domain N-glycans facilitate viral internalization in respiratory epithelial cells.

N-glycosylation plays an important role in the pathogenesis of viral infections. However, the role of SARS-CoV-2 RBD N-glycosylation in viral entry remains elusive. In this study, we expressed and purified N331 and N343 N-glycosite mutants of SARS-CoV-2 RBD. We found that de-glycosylation at N331 and N343 drastically reduces the RBD binding to ACE2. More importantly, based on qualitative and quantitative virology research methods, we show that the mutation of RBD N-glycosites interfered with SARS-CoV-2 internalization rather than attachment potentially by decreasing RBD binding to the receptors. Also, the double N-glycosites mutant (N331 + N343) showed significantly increased sensitivity against the designated RBD neutralizing antibodies. Taken together, these results suggest that N-glycosylation of SARS-CoV-2 RBD is not only critical for viral internalization into respiratory epithelial cells but also shields the virus from neutralization. It may provide new insights into the biological process of early-stage SARS-CoV-2 infection with potential therapeutic implications.

The longitudinal relationship between sleep problems and school burnout in adolescents: A cross-lagged panel analysis.

INTRODUCTION: Despite the growing body of cross-sectional research linking sleep problems and school burnout, hardly any research has investigated the longitudinal relationship between these two constructs. The aim of this study was to examine the bidirectional association between sleep problems and school burnout in middle school students. METHODS: A prospective design was used incorporating four time points (approximately 6-month interval). The participants were 1226 (50.3% girls) middle school students from 4 public schools who were in 7th grade at baseline. On average, participants were approximately 12.5 years old at the beginning of the study (Mage = 12.73, SD = 0.68). All participants completed self-report measures in classrooms during regular school hours. The data were analyzed using a cross-lagged structural equation model. We also examined the stability of sleep problems and school burnout in time, and investigated the moderating role of gender. RESULTS: The results indicated there is a moderate stability for both sleep problems and school burnout, and those students with sleep problems were more likely to develop school burnout, and vice versa. Gender did not moderate the bidirectional relationship. CONCLUSIONS: These results suggest that future studies should investigate whether implementing intervention for sleep problems can decrease the occurrence of school burnout and whether interventions targeting school burnout can improve sleep problems.

A Calibration Method for a Self-Rotating, Linear-Structured-Light Scanning, Three-Dimensional Reconstruction System Based on Plane Constraints.

This paper proposes a calibration method for a self-rotating, linear-structured-light (LSL) scanning, three-dimensional reconstruction system based on plane constraints. The point cloud of plane target collected by the self-rotating, LSL scanning, 3D reconstruction system should be constrained to the basic principle of the plane equation; it can quickly and accurately calibrate the position parameters between the coordinate system of the LSL module and the coordinate system of the self-rotating, LSL scanning, 3D reconstruction system. Additionally, the transformation equation could be established with the calibrated optimal position parameters. This paper obtains the above-mentioned position parameters through experiments and uses the calibrated self-rotating, LSL scanning, 3D reconstruction system to perform three-dimensional scanning and reconstruction of the test piece. The experimental results show that the calibration method can effectively improve the measurement accuracy of the system.

A Novel Transfer Function Based Ring-Down Suppression System for PMUTs.

In this paper, a novel ring-down suppression system based on transfer function is proposed for the first time to suppress the ring-down time and decrease the blind area of PMUTs (Piezoelectric Micromachined Ultrasonic Transducers). This suppression system includes a transfer function and a simple P (proportion) controller, which can reduce the ring-down time without degrading any performances of PMUTs. The transfer function serves as a virtual PMUT device, feeding its output into the P controller; then, the P controller generates a suppression signal to the actual PMUT device. The ring-down time of a 115-kHz PMUT array is demonstrated to be reduced by up to 93% through the suppression system. In addition, the P controller has been experimentally optimized, reducing the blind area of the PMUT array by about 40%. Moreover, a low ring-down PMUTs system design guideline is established, which is practical and straightforward for industrial scenarios. Finally, the system can be easily integrated into ASIC (Application Specific Integrated Circuit).

Magnetic bead-enzyme assemble for triple-parameter telomerase detection at single-cell level.

In this work, we developed a triple-parameter strategy for the detection of telomerase activity from cancer cells and urine samples. This strategy was developed based on magnetic bead-enzyme hybrids combined with fluorescence analysis, colorimetric assay, or adenosine triphosphate (ATP) meter as readout. The application of magnetic bead-enzyme hybrids has the advantages of magnetic separation and signal amplification. These detection methods can be used individually or in combination to achieve the optimal sensing performance and make the results more convincing. Among them, the ATP meter with portable size had easy operation and low cost, and this response strategy provided a higher sensitivity at the single-cell level. The designed strategy was suitable as naked-eye sensor and point-of-care testing (POCT) for rapid assaying of telomerase activity. Graphical abstract Magnetic bead-enzyme assemble for triple-parameter telomerase detection.

Effect of paeonol on proliferation, apoptosis, migration, invasion and glutamine of gastric cancer cells via circSFMBT2/miR-665 axis.

This experiment was performed to investigate the effect of paeonol on the proliferation, apoptosis, migration, invasion and glutamine of gastric cancer HGC-27 cells and its possible mechanism. For this purpose, the MTT method was used to detect cell viability; Flow cytometry experiment was used to detect cell apoptosis; Transwell chamber experiment was used to detect cell migration and invasion; Western blotting was used to detect the expression levels of MMP2 and MMP9 protein; The decomposition of glutamine was evaluated by detecting the expression levels of glutamine, glutamic acid and α-ketoglutarate (α-KG). This study used RT-PCR to detect the expression of circSFMBT2 and miR-665. The targeting relationship between circSFMBT2 and miR-665 was verified by the dual-luciferase report experiment and RIP experiment. Results showed that different concentrations of Paeonol could significantly inhibit the proliferation, migration, invasion and glutamine decomposition of HGC-27 cells, and induce cell apoptosis in a dose-dependent manner. In gastric cancer tissues and cells, the expression of circSFMBT2 was up-regulated, and the expression of miR-665 was down-regulated. Over-expression of circSFMBT2 could partially restore the effects of paeonol on the proliferation, apoptosis, migration, invasion and glutamine of HGC-27 cells. CircSFMBT2 could target and negatively regulate the expression of miR-665. Overexpression of miR-665 could partially restore the effects of Pae and circSFMBT2 on the proliferation, apoptosis, migration, invasion and glutamine of HGC-27 cells. It was concluded that paeonol can inhibit the proliferation, migration, invasion and glutamine decomposition of gastric cancer HGC-27 cells via circSFMBT2/miR-665 axis, and also induce cell apoptosis.

Spatial and seasonal variations of antibiotic resistance genes and antibiotics in the surface waters of Poyang Lake in China.

Antibiotic resistance genes (ARGs) and antibiotics in the aquatic environment raise health concerns particularly on the dispersal and persistence of antibiotic resistance. Large lakes, which serve as catch basins of anthropogenic inputs provide an ideal environment for understanding the occurrence and accumulation of ARGs and antibiotics in freshwater environments. Here, the largest freshwater lake in China, Poyang Lake, located in the developing district of Yangtze valley was used to study the characterization of the spatial and seasonal variation of both ARGs and antibiotics. Results showed that twelve tested ARGs (sul1, sul2, sul3, tetA, tetB, tetC, tetH, tetW, tetO, tetM, qnrS, and qnrB) were detected in the surface waters of Poyang Lake, with a detection frequency ranging from 19.2% to 100%, and sul2 and tetA genes were identified as potential indicators of ARG pollution in this region. Among the 11 analyzed antibiotics, sulfonamides were the predominant antibiotics with a contribution of more than 50% to the total concentrations of tested antibiotics. The total concentrations of both ARGs and antibiotics were higher in the dry season than those in the wet season. Furthermore, ARGs and antibiotics in the surface waters also varied with sampling locations, being consistently at riverine tributaries. Positive correlations were also observed between the concentrations of ARGs and antibiotics, as well as the integron gene (intI1), indicating that antibiotics and intI1 may be playing important roles in the occurrence and dispersal of ARGs in the surface waters. Lastly, our results suggest that intensive anthropogenic activities related to antibiotic usage have substantially contributed to the occurrence and persistence of ARGs and antibiotics in Poyang Lake.

Quantum Dot Nanobeacons for Single RNA Labeling and Imaging.

Detection and imaging RNAs in live cells is in high demand. Methodology for such a purpose is still a challenge, particularly for single RNA detection and imaging in live cells. In this study, a type of quantum dot (QD) nanobeacon with controllable valencies was constructed by precisely conjugating the black hole quencher (BHQ1) and phosphorothioate comodified DNA onto CdTe:Zn2+ QDs via a one-pot hydrothermal method. The nanobeacon with only one conjugated DNA was used to label and detect low-abundance nucleic acids in live cells, and single HIV-1 RNAs were detected and imaged in live HIV-1 integrated cells. Additionally, QD nanobeacon-labeled HIV-1 genomic RNAs were encapsulated in progeny viral particles, which can be used to track the uncoating process of single viruses. The current study provides a platform for nucleic acid labeling and imaging with high sensitivity, being especially meaningful for tracking of individual RNAs in live cells.

iTRAQ-Based Comparative Proteomic Analysis Provides Insights into Molecular Mechanisms of Salt Tolerance in Sugar Beet (Beta vulgaris L.).

Salinity is one of the major abiotic stress factors that limit plant growth and crop yield worldwide. To understand the molecular mechanisms and screen the key proteins in response of sugar beet (Beta vulgaris L.) to salt, in the present study, the proteomics of roots and shoots in three-week-old sugar beet plants exposed to 50 mM NaCl for 72 h was investigated by isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technology. The results showed that 105 and 30 differentially expressed proteins (DEPs) were identified in roots and shoots of salt-treated plants compared with untreated plants, respectively. There were 46 proteins up-regulated and 59 proteins down-regulated in roots; and 13 up-regulated proteins and 17 down-regulated proteins found in shoots, respectively. These DEPs were mainly involved in carbohydrate metabolism, energy metabolism, lipid metabolism, biosynthesis of secondary metabolites, transcription, translation, protein folding, sorting, and degradation as well as transport. It is worth emphasizing that some novel salt-responsive proteins were identified, such as PFK5, MDH, KAT2, ACAD10, CYP51, F3H, TAL, SRPR, ZOG, V-H⁺-ATPase, V-H⁺-PPase, PIPs, TIPs, and tubulin α-2/ß-1 chain. qRT-PCR analysis showed that six of the selected proteins, including BvPIP1-4, BvVP and BvVAP in root and BvTAL, BvURO-D1, and BvZOG in shoot, displayed good correlation between the expression levels of protein and mRNA. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings should significantly improve the understanding of the molecular mechanisms involved in salt tolerance of sugar beet plants.

Live Visualization of HIV-1 Proviral DNA Using a Dual-Color-Labeled CRISPR System.

HIV latency is one of the major problems in HIV/AIDS cure. Imaging single-copy integrated proviral HIV DNA in host cell has both virology and clinical significance but remains technical challenge. Here, we developed a dual-color labeled CRISPR system to image the HIV-1 integrated proviral DNA in latently infected cells. The pair of CRISPRs was fluorescently labeled with two different color QDs using two alternative bioorthogonal ligation reactions. Integrated HIV-sequences are successfully mapped based on the colocalized signals of QDs in living cells. Compared to the existing zinc finger proteins and TALENs, the CRISPR system is much easier to operate and more efficient in imaging of internal genomic loci. Therefore, the proposed method could be not only a powerful tool for imaging proviral HIV-1, but also a versatile platform to image single genomic loci in living cells.

High-quality Mach-Zehnder interferometer based on a microcavity in single-multi-single mode fiber structure for refractive index sensing.

A fiber inline Mach-Zehnder interferometer (MZI) based on a microcavity with two symmetric openings in single-multi-single mode fiber (SMSF) structure is proposed. By using the finite difference beam propagation method (FD-BPM), the interference spectrum simulation result shows that the MZI can still have high-quality interference even if the microcavity deviates along the radial direction for 3 µm. Therefore, it allows a larger fabrication tolerance and tremendously decreases the fabrication difficulty. Then a microcavity with two symmetric openings in SMSF was fabricated by using femtosecond laser-induced water breakdown. The insertion loss of the microcavity immerged in water is only -8 dB, and the MZ interference peak contrast in the transmission spectrum reaches more than 30 dB. The MZI based on the microcavity in SMSF can be used as a practical liquid refractive index sensor as its high-quality interference spectrum, ultrahigh sensitivity (9756.75 nm/RIU), high refractive index resolution (2×10-5 RIU), good linearity (99.93%), and low-temperature crosstalk (0.04 nm/°C).

Activation of PI3K/Akt pathway by CD133-p85 interaction promotes tumorigenic capacity of glioma stem cells.

The biological significance of a known normal and cancer stem cell marker CD133 remains elusive. We now demonstrate that the phosphorylation of tyrosine-828 residue in CD133 C-terminal cytoplasmic domain mediates direct interaction between CD133 and phosphoinositide 3-kinase (PI3K) 85 kDa regulatory subunit (p85), resulting in preferential activation of PI3K/protein kinase B (Akt) pathway in glioma stem cell (GSC) relative to matched nonstem cell. CD133 knockdown potently inhibits the activity of PI3K/Akt pathway with an accompanying reduction in the self-renewal and tumorigenicity of GSC. The inhibitory effects of CD133 knockdown could be completely rescued by expression of WT CD133, but not its p85-binding deficient Y828F mutant. Analysis of glioma samples reveals that CD133 Y828 phosphorylation level is correlated with histopathological grade and overlaps with Akt activation. Our results identify the CD133/PI3K/Akt signaling axis, exploring the fundamental role of CD133 in glioma stem cell behavior.

ZxNHX controls Na⁺ and K⁺ homeostasis at the whole-plant level in Zygophyllum xanthoxylum through feedback regulation of the expression of genes involved in their transport.

BACKGROUND AND AIMS: In order to cope with arid environments, the xerohalophyte Zygophyllum xanthoxylum efficiently compartmentalizes Na(+) into vacuoles, mediated by ZxNHX, and maintains stability of K(+) in its leaves. However, the function of ZxNHX in controlling Na(+) and K(+) homeostasis at the whole-plant level remains unclear. In this study, the role of ZxNHX in regulating the expression of genes involved in Na(+) and K(+) transport and spatial distribution was investigated. METHODS: The role of ZxNHX in maintaining Na(+) and K(+) homeostasis in Z. xanthoxylum was studied using post-transcriptional gene silencing via  Agrobacterium-mediated transformation. Transformed plants were grown with or without 50 mm NaCl, and expression levels and physiological parameters were measured. KEY RESULTS: It was found that 50 mm NaCl induced a 620 % increase in transcripts of ZxSOS1 but only an 80 % increase in transcripts of ZxHKT1;1 in roots of wild-type (WT) plants. Consequently, the ability of ZxSOS1 to transport Na(+) exceeded that of ZxHKT1;1, and Na(+) was loaded into the xylem by ZxSOS1 and delivered to the shoots. However, in a ZxNHX-silenced line (L7), the capacity to sequester Na(+) into vacuoles of leaves was weakened, which in turn regulated long-distance Na(+) transport from roots to shoots. In roots of L7, NaCl (50 mm) increased transcripts of ZxSOS1 by only 10 %, whereas transcripts of ZxHKT1;1 increased by 53 %. Thus, in L7, the transport ability of ZxHKT1;1 for Na(+) outweighed that of ZxSOS1. Na(+) was unloaded from the xylem stream, consequently reducing Na(+) accumulation and relative distribution in leaves, but increasing the relative distribution of Na(+) in roots and the net selective transport capacity for K(+) over Na(+) from roots to shoots compared with the WT. Silencing of ZxNHX also triggered a downregulation of  ZxAKT1 and ZxSKOR in roots, resulting in a significant decrease in K(+) accumulation in all the tissues in plants grown in 50 mm NaCl. These changes led to a significant reduction in osmotic adjustment, and thus an inhibition of growth in ZxNHX-silenced lines. CONCLUSIONS: The results suggest that ZxNHX is essential for controlling Na(+), K(+) uptake, long-distance transport and their homeostasis at whole-plant level via feedback regulation of the expression of genes involved in Na(+), K(+) transport. The net result is the maintenance of the characteristic salt accumulation observed in Z. xanthoxylum and the regulation of its normal growth. A model is proposed for the role of ZxNHX in regulating the Na(+) transport system in Z. xanthoxylum under saline conditions.

[Functional mechanisms of WRKY transcription factors in regulating plant response to abiotic stresses].

WRKYs is a unique family of transcription factors (TFs) in plants, and belongs to the typical multifunctional regulator. It is involved in the regulation of multiple signaling pathways. This type of transcription factor is characterized to contain about 60 highly conservative amino acids as the WRKY domain, and usually also has the Cys2His2 or Cys2His-Cys zinc finger structure. WRKYs can directly bind to the W-box sequence ((T)(T) TGAC (C/T)) in the promoter region of the downstream target gene, and activate or inhibit the transcription of the target genes by interacting with the target protein. They may up-regulate the expression of stress-related genes through integrating signal pathways mediated by abscisic acid (ABA) and reactive oxygen species (ROS), thus playing a vital role in regulating plant response to abiotic stresses. This review summarizes the advances in research on the structure and classification, regulatory approach of WRKYs, and the molecular mechanisms of WRKYs involved in response to drought and salt stresses, and prospects future research directions, with the aim to provide a theoretical support for the genetic improvement of crop in response to abiotic stresses.