Label-Free Direct Identification of MicroRNAs Based on a Narrow Constant-Inner-Diameter Emitter Mass Spectrometry Analysis.

MicroRNAs (miRNAs) are a class of endogenous noncoding small RNAs that play important roles in various biological processes and diseases. Direct determination of miRNAs is a cost-efficient and accurate method for analysis. Herein, we established a novel method for the analysis of miRNAs based on a narrow constant-inner-diameter mass spectrometry emitter. We utilized the gravity-assisted sleeving etching method to prepare a constant-inner-diameter mass spectrometry emitter with a capillary inner diameter of 5.5 µm, coupled it with a high-voltage power supply and a high-resolution mass spectrometer, and used it for miRNA direct detection. The method showed high sensitivity and reproducibility for the analysis of four miRNAs, with a limit of detection of 100 nmol/L (170 amol) for the Hsa-miR-1290 analysis. Compared with commercial ion sources, our method achieved higher sensitivity for miRNA detection. In addition, we analyzed the total miRNAs in the A549 cells. The result indicated that both spiked and endogenous miRNAs could be quantified with high accuracy. As a result, this method offers a promising platform for highly sensitive and accurate miRNA analysis. Furthermore, this approach can be extended to the analysis of other small oligonucleotides and holds the potential for studying clinical samples and facilitating disease diagnosis.

Establishment of a diagnostic model of endometriosis based on disulfidptosis-related genes.

OBJECTIVES: We aimed to establish a diagnostic model of endometriosis (EM) based on disulfidptosis-related genes (DRGs). MATERIALS AND METHODS: The mRNA expression data of EM were downloaded from the gene expression omnibus database and subjected to differential analysis, and co-expression analysis was performed based on 10 disulfidptosis genes to acquire DRGs. The differentially expressed DRGs were subjected to biofunctional analysis. Lasso analysis and support vector machine-recursive feature elimination (SVM-RFE) analysis were employed to extract the intersection of feature genes as biomarkers, and the diagnostic values of biomarkers for EM were evaluated based on receiver operating characteristic curves. The correlations between biomarkers and the immune microenvironment were assessed by Pearson analysis of biomarkers and immune cell infiltration levels. RESULTS: Transforming growth factor ß stimulated protein clone 22 domain family member 4 (TSC22D4), and F-box/SPRY domain-containing protein 1 (FBXO45) worked as the diagnostic classifiers in EM, with an obvious decrease in FBXO45 expression and an evident increase in TSC22D4 expression. The areas under the curves of FBXO45 and TSC22D4 were 0.752 and 0.706, respectively, and the area of FBXO45 combined with TSC22D4 reached 0.865, suggesting that TSC22D4 and FBXO45 had high predictive values. The diagnostic markers were closely correlated with immune cell infiltration. CONCLUSION: The diagnostic markers constructed based on disulfidptosis are good predictors for EM, which have close correlations with EM.

Single-Cell Metabolomics-Based Strategy for Studying the Mechanisms of Drug Action.

Studying the mechanisms of drug antitumor activity at the single-cell level can provide information about the responses of cell subpopulations to drug therapy, which is essential for the accurate treatment of cancer. Due to the small size of single cells and the low contents of metabolites, metabolomics-based approaches to studying the mechanisms of drug action at the single-cell level are lacking. Herein, we develop a label-free platform for studying the mechanisms of drug action based on single-cell metabolomics (sMDA-scM) by integrating intact living-cell electro-launching ionization mass spectrometry (ILCEI-MS) with metabolomics analysis. Using this platform, we reveal that non-small-cell lung cancer (NSCLC) cells treated by gefitinib can be clustered into two cell subpopulations with different metabolic responses. The glutathione metabolic pathway of the subpopulation containing 14.4% of the cells is not significantly affected by gefitinib, exhibiting certain resistance characteristics. The presence of these cells masked the judgment of whether cysteine and methionine metabolic pathway was remarkably influenced in the analysis of overall average results, revealing the heterogeneity of the response of single NSCLC cells to gefitinib treatment. The findings provide a basis for evaluating the early therapeutic effects of clinical medicines and insights for overcoming drug resistance in NSCLC subpopulations.

Sensitive electrochemical measurement of nitric oxide released from living cells based on dealloyed PtBi alloy nanoparticles.

Nitric oxide (NO), as a vital signaling molecule related to different physiological and pathological processes in living systems, is closely associated with cancer and cardiovascular disease. However, the detection of NO in real-time remains a difficulty. Here, PtBi alloy nanoparticles (NPs) were synthesized, dealloyed, and then fabricated to NP-based electrodes for the electrochemical detection of NO. Transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and nitrogen physical adsorption/desorption show that dealloyed PtBi alloy nanoparticles (dPtBi NPs) have a porous nanostructure. Electrochemical impedance spectroscopy and cyclic voltammetry results exhibit that the dPtBi NP electrode possesses unique electrocatalytic features such as low charge transfer resistance and large electrochemically active surface area, which lead to its excellent NO electrochemical sensing performance. Owing to the higher density of catalytical active sites formed PtBi bimetallic interface, the dPtBi NP electrode displays superior electrocatalytic activity toward the oxidation of NO with a peak potential at 0.74 V vs. SCE. The dPtBi NP electrode shows a wide dynamic range (0.09-31.5 µM) and a low detection limit of 1 nM (3σ/k) as well as high sensitivity (130 and 36.5 µA µM-1 cm-2). Moreover, the developed dPtBi NP-based electrochemical sensor also exhibited good reproducibility (RSD 5.7%) and repeatability (RSD 3.4%). The electrochemical sensor was successfully used for the sensitive detection of NO produced by live cells. This study indicates a highly effective approach for regulating the composition and nanostructures of metal alloy nanomaterials, which might provide new technical insights for developing high-performance NO-sensitive systems, and have important implications in enabling real-time detection of NO produced by live cells.

Diagnostic value of combined pelvic floor ultrasound parameters for pelvic floor dysfunction and risk factors.

OBJECTIVE: To investigate the diagnostic value of pelvic floor ultrasound parameters in combination for pelvic floor dysfunction (PFD), and to explore the risk factors. METHODS: Forty PFD patients treated from April2019to December 2020(case group) and another 40 healthy women (control group) were enrolled. Their clinical data were collected, and both groups received three-dimensional (3D) ultrasound of the pelvic floor. The diagnostic value of pelvic floor ultrasound parameters for PFD was assessed by receiver operating characteristic (ROC) curves. The risk factors of PFD were evaluated by multivariate logistic regression analysis. RESULTS: The area under the ROC curve (AUC), sensitivity, and specificity of the parameters in combination for predicting PFD were 0.851 [95% confidence interval (CI): 0.743-0.959], 0.901, and 0.812, respectively, indicating acceptable accuracy. Results of logistic regression analysis revealed that spontaneous delivery, lateral episiotomy/laceration, and large bladder neck rotation angle, posterior urethrovesical angle (PUA), bladder neck tilt angle, bladder neck distance (BND), levator hiatus area (LHA) (at anal contraction), R-LHA and V-LHA were risk factors for PFD (p < 0.05), while physical exercise was a protective factor (p < 0.05). ROC curve analysis revealed that the AUC, sensitivity, and specificity of the forest map model were 0.822 (95% CI: 0.759-0.885), 0.942, and 0.601, respectively, indicating acceptable accuracy of the model. Internal data validation of the model demonstrated consistence of the predicted occurrence of PFD with the actual one. CONCLUSIONS: Spontaneous delivery, lateral episiotomy/laceration, and large bladder neck rotation angle, PUA, bladder neck tilt angle, BND, LHA (at anal contraction), R-LHA and V-LHA were risk factors for PFD.

Hydrogen-rich water alleviates chilling injury-induced lignification of kiwifruit by inhibiting peroxidase activity and improving antioxidant system.

BACKGROUND: Kiwifruit is prone to chilling stress and displays chilling injury (CI) such as lignification; however, the underlying physicochemical mechanism remains largely unknown. Here, the changes in levels of quality attributes, lignin biosynthesis, antioxidant system and sugars were compared in kiwifruit between control and hydrogen-rich water (HRW) treatments during cold storage for 90 days at 0 °C. RESULTS: The results reveal that HRW is an effective measure for CI alleviation, as indicated by the decrease in lignification level with repressed peroxidase activity but enhanced polyphenol oxidase activity. The amelioration of membrane peroxidation was suggested by the repressed levels of H2 O2 and malondialdehyde. They were accompanied by the improvement of antioxidant system, which is supported by the enhancement of sugars including fructose and glucose. CONCLUSION: In conclusion, HRW can enhance chilling tolerance, as suggested by the alleviation of lignification through inhibiting peroxidase activity and elevating the antioxidant system to attenuate membrane peroxidation. © 2022 Society of Chemical Industry.

Biomimetic Nanocomposites Camouflaged with Hybrid Cell Membranes for Accurate Therapy of Early-Stage Glioma.

Glioma features high fatality rate and short survival time of patients due to its fast growth speed and high invasiveness, hence timely treatment of early-stage glioma is extremely important. However, the blood brain barrier (BBB) severely prevents therapeutic agents from entering the brain; meanwhile, the non-targeted distribution of agents always causes side effects to vulnerable cerebral tissues. Therefore, delivery systems that possess both BBB penetrability and precise glioma targeting ability are keenly desired. We herein proposed a hybrid cell membrane (HM) camouflage strategy to construct therapeutic nanocomposites, in which HM consisting of brain metastatic breast cancer cell membrane and glioma cell membrane was prepared with a simple membrane fusion pathway. By coating HM onto drug-loaded nanoparticles, the as-obtained biomimetic therapeutic agent (termed HMGINPs) inherited satisfying BBB penetrability and homologous glioma targeting ability simultaneously from the two source cells. HMGINPs exhibited good biocompatibility and superior therapeutic efficacy towards early-stage glioma.

Attomole-Level Multiplexed Detection of Neurochemicals in Picoliter Droplets by On-Chip Nanoelectrospray Ionization Coupled to Mass Spectrometry.

While droplet microfluidics is becoming an effective tool for biomedical research, sensitive detection of droplet content is still challenging, especially for multiplexed analytes compartmentalized within ultrasmall droplets down to picoliter volumes. To enable such measurements, we demonstrate a silicon-based integrated microfluidic platform for multiplexed analysis of neurochemicals in picoliter droplets via nanoelectrospray ionization (nESI)-mass spectrometry (MS). An integrated silicon microfluidic chip comprising downscaled 7 µm-radius channels, a compact T-junction for droplet generation, and an integrated nESI emitter tip is used for segmentation of analytes into picoliter compartments and their efficient delivery for subsequent MS detection. The developed system demonstrates effective detection of multiple neurochemicals encapsulated within oil-isolated plugs down to low picoliter volumes. Quantitative measurements for each neurochemical demonstrate limits of detection at the attomole level. Such results are promising for applications involving label-free and small-volume detection for monitoring a range of brain chemicals.

A π-Conjugated Polyimide-Based High-Performance Aqueous Potassium-Ion Asymmetric Supercapacitor.

Aqueous asymmetric supercapacitor has captured widespread attention as a sustainable high-power energy resource. Organic electrode materials are appealing owing to their sustainability and high redox reactivity, but suffer from structural instability and low power density. Here the π-conjugated polyimide-based organic electrodes with different lengths of alkyl chains are explored to achieve high rate capability and long lifespan in an aqueous K+ -ion electrolyte. The fabricated asymmetric supercapacitor exhibits high capacities of 107 mAh g-1 at 2 A g-1 and 67 mAh g-1 at 90 A g-1 . A specific capacity of 65 mAh g-1 over 70% of the initial performance is obtained after 65 000 cycles. Molecular engineering of long alkyl chains in polyimide can reduce the degree of π-conjugation and spatially block the π-conjugated imide bond with limited redox activity but improved stability against chemical degradation. Further electrochemical quartz crystal microbalance, ex-situ Fourier transformed infrared spectroscopy, and X-ray photoelectron spectroscopy characterizations reveal the pseudocapacitance behavior originating from the π-conjugated polyimide-based redox reaction with potassium ions and hydrated potassium ions. A promising polyimide-based polymer with extended π-conjugated system for high-performance asymmetric supercapacitor is showcased.

Jasmonic acid and salicylic acid induce the accumulation of sucrose and increase resistance to chilling injury in peach fruit.

BACKGROUND: Salicylic acid (SA) and jasmonic acid (JA) can both enhance resistance of chilling injury (CI) in cold-storage peach fruit, but the regulatory mechanisms involved and whether there is a coordinated regulation between them is unclear. In this study, postharvest peach fruit were treated with an aqueous SA solution for 15 min or an aqueous JA solution for 30 s before storage at 4 °C for 35 days. RESULTS: SA and JA treatments both delayed and reduced development of internal browning (a symptom of CI) and induced the accumulation of hydrogen peroxide and sucrose. The SA and JA also reduced catalase and peroxidase activities, which are involved in hydrogen peroxide generation. The SA and JA treatments significantly regulated the transcript abundance of genes related to sucrose biosynthesis and degradation consistent with the observed increase in sucrose content. CONCLUSION: These results intimate that JA and SA may be involved in coordinating the alleviation of CI via increased accumulation of sucrose. © 2021 Society of Chemical Industry.

Separating and Profiling Phosphatidylcholines and Triglycerides from Single Cellular Lipid Droplet by In-Tip Solvent Microextraction Mass Spectrometry.

The analysis of lipid droplets (LDs) by mass spectrometry at the single LD level is still an analytical challenge. In this work, we developed a novel technique termed in-tip solvent microextraction mass spectrometry for the separation and profiling of phosphatidylcholines and triglycerides within a single LD. This method has been successfully used to analyze LDs in mammalian cells and to compare the profiles of triglycerides and phosphatidylcholines in LDs induced at different conditions. Our method has the potential to be applied to such fields as fundamental lipid biology to further our understanding on the mechanisms of lipid production, lipid packaging, and their pathophysiological roles.

Rapid Analysis of Unsaturated Fatty Acids on Paper-Based Analytical Devices via Online Epoxidation and Ambient Mass Spectrometry.

In this work, we demonstrate a novel design that allows rapid online identification and quantitation of unsaturated fatty acid C═C location isomers via epoxidation and ambient mass spectrometry (MS). Unsaturated fatty acid solution was loaded on a paper strip placed between a low-temperature plasma probe and the inlet of a mass spectrometer. Reactive oxygen species in the plasma promoted epoxidation at the C═C, and the product was simultaneously ionized. Upon collision-induced dissociation (CID), the epoxidation product was fragmented to release diagnostic ions specific to the C═C location. The whole analytical workflow can be completed within 5 s and is particularly promising for point-of-care (POC) clinical diagnosis, considering its fast, high-throughput nature, and coupling with paper-based analytical devices.

FaMYB44.2, a transcriptional repressor, negatively regulates sucrose accumulation in strawberry receptacles through interplay with FaMYB10.

Sugar and acid metabolism are critical for fruit ripening and quality formation, but the underlying regulatory mechanisms are largely unknown. Here, we identified a transcriptional repressor, FaMYB44.2, that regulates sugar and acid accumulation in strawberry (Fragaria × ananassa 'Benihoppe') receptacles. We transiently expressed FaMYB44.2 in strawberry fruit and conducted metabolic and molecular analyses to explore the role of FaMYB44.2 in sugar and acid accumulation in strawberry. We found that FaMYB44.2 negatively regulates soluble sugar accumulation and malic acid content and represses the expression of numerous structural genes, including FaSPS3, a key gene in sucrose accumulation. From the white fruit stage onwards, the repressive effect of FaMYB44.2 on FaSPS3 is reversed by FaMYB10, which positively regulates anthocyanin accumulation. Our results indicate that FaMYB10 suppresses FaMYB44.2 expression; weakens the interaction between FaMYB44.2 and its co-repressor, FabHLH3; and cooperates with FabHLH3 to activate the expression of FaSPS3. The interplay between FaMYB10 and FaMYB44.2 results in sucrose accumulation in ripe strawberry fruits. In addition, the repressive effect of FaMYB44.2 on sucrose accumulation is enhanced by jasmonic acid. This study provides new insights into the regulatory mechanisms of sucrose accumulation and sheds light on the interplay between regulatory proteins during strawberry fruit ripening and quality formation.

Identification and Quantitation of C═C Location Isomers of Unsaturated Fatty Acids by Epoxidation Reaction and Tandem Mass Spectrometry.

Unsaturated fatty acids (FAs) serve as nutrients, energy sources, and signaling molecules for organisms, which are the major components for a large variety of lipids. However, structural characterization and quantitation of unsaturated FAs by mass spectrometry remain an analytical challenge. Here, we report the coupling of epoxidation reaction of the C═C in unsaturated FAs and tandem mass spectrometry (MS) for rapid and accurate identification and quantitation of C═C isomers of FAs in a shotgun lipidomics approach. Epoxidation of the C═C leads to the production of an epoxide which, upon collision induced dissociation (CID), produces abundant diagnostic ions indicative of the C═C location. The total intensity of the same set of diagnostic ions for one specific FA C═C isomer was also used for its relative and absolute quantitation. The simple experimental setup, rapid reaction kinetics (<2 min), high reaction yield (>90% for monounsaturated FAs), and easy-to-interpret tandem MS spectra enable a promising methodology particularly for the analysis of unsaturated FAs in complex biological samples such as human plasma and animal tissues.

Profiling of cardiolipins and their hydroperoxides in HepG2 cells by LC/MS.

Cardiolipin (CL) exists as crucial functional phospholipid in mitochondria. The oxidation of CL is concerned with mitochondrial dysfunction and various diseases. As main oxidation products, CL hydroperoxide (CL-OOH) plays a key role in intermediating oxidative reaction. Thus, direct analysis of CL-OOH is of great interest. In the present study, CL and CL-OOH profiles were analyzed in oxidized HepG2 cell lipid via HPLC-Orbitrap MS/MS. Furthermore, the contents of individual molecular species were compared between intact and AAPH-oxidized HepG2 cells. In total, 46 CL and 18 CL-OOH were identified from oxidized cell lipids, while 21 CL and 9 CL-OOH were detected in AAPH-treated cells. Most CL depleted significantly after AAPH inducement, with percentages varying from 8.3% (CL70:7) to 73.7% (CL72:4), depending on fatty acyl composition. While almost all the CL-OOH remarkably increased, among them 68:6-, 72:6-, and 72:7-OOHs were only detected in AAPH-treated cells. CL68:5- and CL68:4-OOH were the most abundant species, while CL70:5-OOH among all the species expressed the highest oxidation percentage of the corresponding CL. Our results showed practical separation, identification, and semi-quantitation of CL-OOH species, which could contribute to approaches to lipidomic analysis of CL and CL-OOH, as well as tracing biomarkers in mitochondrial oxidative stress diagnosis. Graphical abstract Illustration represents cardiolipin hydroperoxide structure and its content increasing in AAPH-treated HepG2 cells by LC/MS analysis.

In Situ Ion-Transmission Mass Spectrometry for Paper-Based Analytical Devices.

Current detection methods for paper-based analytical devices (PADs) rely on spectroscopic and electrochemical properties, which place special requirements on the analyte or need analyte labeling. Here, ion-transmission mass spectrometry (MS) was proposed for coupling with PADs to enable rapid in situ MS analysis of the sample on paper. The sample was analyzed directly on paper via analyte ionization by ions transmitted through the paper, generated by a low-temperature plasma probe. Prior to MS analysis, the sample can be separated by paper electrophoresis or by paper chromatography, among a variety of other features offered by PADs. The versatility of this technique was demonstrated by MS analysis of a paper microarray, a mixture of amino acids, and whole blood doped with drugs on PADs.

Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy.

We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.

Coupling a solid phase microextraction (SPME) probe with ambient MS for rapid enrichment and detection of phosphopeptides in biological samples.

In this study, we developed a probe-electrospray ionization method by coupling a SPME probe modified with nanosized TiO2 directly to nanoESI-MS for the phosphoproteome analysis, which demonstrated excellent selectivity and sensitivity for enrichment of phosphopeptides in complex biological samples.

Single cell analysis with probe ESI-mass spectrometry: detection of metabolites at cellular and subcellular levels.

Molecular analysis at cellular and subcellular levels, whether on selected molecules or at the metabolomics scale, is still a challenge now. Here we propose a method based on probe ESI mass spectrometry (PESI-MS) for single cell analysis. Detection of metabolites at cellular and subcellular levels was successfully achieved. In our work, tungsten probes with a tip diameter of about 1 µm were directly inserted into live cells to enrich metabolites. Then the enriched metabolites were directly desorbed/ionized from the tip of the probe for mass spectrometry (MS) detection. The direct desorption/ionization of the enriched metabolites from the tip of the probe greatly improved the sensitivity by a factor of about 30 fold compared to those methods that eluted the enriched analytes into a liquid phase for subsequent MS detection. We applied the PESI-MS to the detection of metabolites in single Allium cepa cells. Different kinds of metabolites, including 6 fructans, 4 lipids, and 8 flavone derivatives in single cells, have been successfully detected. Significant metabolite diversity was observed among different cells types of A. cepa bulb and different subcellular compartments of the same cell. We found that the inner epidermal cells had about 20 fold more fructans than the outer epidermal cells, while the outer epidermal cells had more lipids. We expected that PESI-MS might be a candidate in the future studies of single cell "omics".

A hierarchical carbon foam-hosted Co2P nanoparticles monolithic electrode for ampere-level and super-durable electrocatalytic hydrogen production.

In this work, we develop a hierarchical carbon foam-based monolithic electrode (Co2P@HCF) from Co2+-adsorbed polyvinyl alcohol (PVA) sponge via the successive carbonization and phosphorization. Owing to the 3D hierarchical porous structure, excellent electrolyte wettability, good mechanical strength, and intimate embedding of highly dispersed Co2P nanoparticles, the Co2P@HCF electrode delivers a high current density of 1.0 A cm-2 for the hydrogen evolution reaction (HER) at ultralow overpotentials of 189.6 and 218.6 mV in 0.5 M H2SO4 and 1.0 M KOH solutions, respectively, with remarkable durability for 100 h.