(+)-Borneol Protects Dopaminergic Neuronal Loss in Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Parkinson's Disease Mice: A Study of Dopamine Level using In Vivo Brain Microdialysis.

Considerable research efforts have been directed toward the symptom relief of Parkinson's disease (PD) by attenuating dopamine (DA) depletion. One common feature of these existing therapies is their unavailability of preventing the neurodegenerative process of dopaminergic neurons. (+)-Borneol, a natural highly lipid-soluble bicyclic monoterpene, has been reported to regulate the levels of monoamine neurotransmitters in the central nervous system and exhibit neuroprotective effects. However, the effect of (+)-borneol on the dopaminergic neuronal loss of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice is not defined. Herein, we first report that 30 mg/kg (+)-borneol significantly attenuated the motor deficits of PD mice, which benefits from markedly increasing the level of DA and decreasing the metabolic rate of DA in the striatum of conscious and freely moving mouse detected by ultraperformance liquid chromatography tandem mass spectrometry online combined with in vivo brain microdialysis sampling. It is worth noting that the enhanced level of DA by (+)-borneol was enabled by the reduction in loss of tyrosine hydroxylase-immunoreactive dopaminergic neurons in the substantia nigra and striatum and promotion of reserpine- or nomifensine-induced DA release in PD mice. Interestingly, (+)-borneol evidently inhibited the decreased expression levels of DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) on the MPTP mouse model of PD. Moreover, (+)-borneol suppressed the neuroinflammation by inhibiting the production of IL-1ß, IL-6, and TNF-α and attenuated oxidative stress by decreasing the level of MDA and increasing the activities of SOD and GSH-px in PD mice. These findings demonstrate that (+)-borneol protects DA neurons by inhibiting neuroinflammation and oxidative stress. Further research work for the neuroprotection mechanism of (+)-borneol will focus on reactive oxygen species-mediated apoptosis. Therefore, (+)-borneol is a potential therapeutic candidate for retarding the neurodegenerative process of PD.

Common neural dysfunction of economic decision-making across psychiatric conditions.

Adaptive decision-making, which is often impaired in various psychiatric conditions, is essential for well-being. Recent evidence has indicated that decision-making capacity in multiple tasks could be accounted for by latent dimensions, enlightening the question of whether there is a common disruption of brain networks in economic decision-making across psychiatric conditions. Here, we addressed the issue by combining activation/lesion network mapping analyses with a transdiagnostic brain imaging meta-analysis. Our findings indicate that there were transdiagnostic alterations in the thalamus and ventral striatum during the decision or outcome stage of decision-making. The identified regions represent key nodes in a large-scale network, which is composed of multiple heterogeneous brain regions and plays a causal role in motivational functioning. The findings suggest that disturbances in the network associated with emotion- and reward-related processing play a key role in dysfunctions of decision-making observed in various psychiatric conditions. This study provides the first meta-analytic evidence of common neural alterations linked to deficits in economic decision-making.

Planktonic eukaryotes in the Chesapeake Bay: contrasting responses of abundant and rare taxa to estuarine gradients.

Phytoplankton are important drivers of aquatic ecosystem function and environmental health. Their community compositions and distributions are directly impacted by environmental processes and human activities, including in the largest estuary in North America, the Chesapeake Bay. It is crucial to uncover how planktonic eukaryotes play fundamental roles as primary producers and trophic links and sustain estuarine ecosystems. In this study, we investigated the detailed community structure and spatiotemporal variations of planktonic eukaryotes in the Chesapeake Bay across space and time for three consecutive years. A clear seasonal and spatial shift of total, abundant, and rare planktonic eukaryotes was evident, and the pattern recurred interannually. Multiple harmful algal species have been identified in the Bay with varied distribution patterns, such as Karlodinium, Heterosigma akashiwo, Protoperidinium sp., etc. Compared to abundant taxa, rare subcommunities were more sensitive to environmental disturbance in terms of richness, diversity, and distribution. The combined effects of temporal variation (13.3%), nutrient availability (10.0%), and spatial gradients (8.8%) structured the distribution of eukaryotic microbial communities in the Bay. Similar spatiotemporal patterns between planktonic prokaryotes and eukaryotes suggest common mechanisms of adjustment, replacement, and species interaction for planktonic microbiomes under strong estuarine gradients. To our best knowledge, this work represents the first systematic study on planktonic eukaryotes in the Bay. A comprehensive view of the distribution of planktonic microbiomes and their interactions with environmental processes is critical in understanding the underlying microbial mechanisms involved in maintaining the stability, function, and environmental health of estuarine ecosystems. IMPORTANCE: Deep sequencing analysis of planktonic eukaryotes in the Chesapeake Bay reveals high community diversity with many newly recognized phytoplankton taxa. The Chesapeake Bay planktonic eukaryotes show distinct seasonal and spatial variability, with recurring annual patterns of total, abundant, and rare groups. Rare taxa mainly contribute to eukaryotic diversity compared to abundant groups, and they are more sensitive to spatiotemporal variations and environmental filtering. Temporal variations, nutrient availability, and spatial gradients significantly affect the distribution of eukaryotic microbial communities. Similar spatiotemporal patterns in prokaryotes and eukaryotes suggest common mechanisms of adjustment, substitution, and species interactions in planktonic microbiomes under strong estuarine gradients. Interannually recurring patterns demonstrate that diverse eukaryotic taxa have well adapted to the estuarine environment with a long residence time. Further investigations of how human activities impact estuarine planktonic eukaryotes are critical in understanding their essential ecosystem roles and in maintaining environmental safety and public health.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

Electric-Field-Induced Selective Directed Transport of Diverse Droplets.

Droplet directional transport is one of the central topics in microfluidics and lab-on-a-chip applications. Selective transport of diverse droplets, particularly in another liquid phase environment with controlled directions, is still challenging. In this work, we propose an electric-field gradient-driven droplet directional transport platform facilitated by a robust lubricant surface. On the platform, we clearly demonstrated a liquid-inherent critical frequency-dominated selective transport of diverse droplets and a driving mechanism transition from electrowetting to liquid dielectrophoresis. Enlightened by the Kelvin-Helmholtz theory, we first realize the directional droplet transport in another liquid phase whenever a permittivity difference exists. Co-transport of multiple droplets and various combinations of droplet types, as well as multifunctional droplet transport modes, are realized based on the presented powerful electric-field gradient-driven platform, overcoming the limitations of the surrounding environment, liquid conductivity, and intrinsic solid-liquid wetting property existing in traditional droplet transport strategies. This work may inspire new applications in liquid separation, multiphase microfluidic manipulation, chemical reagent selection, and so on.

Precisely Activating cGAS-STING Pathway with a Novel Peptide-Based Nanoagonist to Potentiate Immune Checkpoint Blockade Cancer Immunotherapy.

As an essential intracellular immune activation pathway, the cGAS-STING pathway has attracted broad attention in cancer treatment. However, low bioavailability, nonspecificity, and adverse effects of small molecule STING agonists severely limit their therapeutic efficacy and in vivo application. In this study, a peptide-based STING agonist is first proposed, and KLA is screened out to activate the cGAS-STING pathway by promoting mitochondrial DNA (mtDNA) leakage. To precisely activate the cGAS-STING pathway and block the PD-1/PD-L1 pathway, a multi-stimuli activatable peptide nanodrug (MAPN) is developed for the effective delivery of KLA and PD-L1 antagonist peptide (CVR). With rational design, MAPN achieved the site-specific release of KLA and CVR in response to multiple endogenous stimuli, simultaneously activating the cGAS-STING pathway and blocking PD-1/PD-L1 pathway, ultimately initiating robust and durable T cell anti-tumor immunity with a tumor growth inhibition rate of 78% and extending the median survival time of B16F10 tumor-bearing mice to 40 days. Overall, antimicrobial peptides, which can promote mtDNA leakage through damaging mitochondrial membranes, may be potential alternatives for small molecule STING agonists and giving a new insight for the design of novel STING agonists. Furthermore, MAPN presents a universal delivery platform for the effective synergy of multiple peptides.

Depth shapes microbiome assembly and network stability in the Mariana Trench.

IMPORTANCE: Exploring microbial interactions and their stability/resilience from the surface to the hadal ocean is critical for further understanding of the microbiome structure and ecosystem function in the Mariana Trench. Vertical gradients did not destabilize microbial communities after long-term evolution and adaption. The uniform niche breadth, diversity, community complexity, and stability of microbiomes in both upper bathypelagic and hadal waters suggest the consistent roles of microbiomes in elemental cycling and adaptive strategies to overcome extreme environmental conditions. Compared with microeukaryotes, bacteria and archaea play a pivotal role in shaping the stability of the hadal microbiome. The consistent co-occurrence stability of microbiomes across vertical gradients was observed in the Mariana Trench. These results illuminate a key principle of microbiomes inhabiting the deepest trench: although distinct microbial communities occupy specific habitats, the interactions within microbial communities remain consistently stable from the upper bathypelagic to the hadal waters.

Environmental gradients shape microbiome assembly and stability in the East China sea.

Microbiomes play a key role in marine ecosystem functioning and sustainability. Their organization and stability in coastal areas, particularly in anthropogenic-influenced regions, however, remains unclear compared with an understanding of how microbial community shifts respond to marine environmental gradients. Here, the assembly and community associations across vertical and horizontal gradients in the East China Sea are systematically researched. The seawater microbial communities possessed higher robustness and lower fragmentation and vulnerability compared to the sediment microbiomes. Spatial gradients act as a deterministic filtering factor for microbiome organization. Microbial communities had lower phylogenetic distance and higher niche breadth in the nearshore and offshore areas compared to intermediate areas. The phylogenetic distance of microbiomes decreased from the surface to the bottom but the niche breadth was enhanced in surface and bottom environments. Vertical gradients destabilized microbial associations, while the community diversity was enhanced. Multivariate regression tree analysis and canonical correspondence analysis indicated that depth, distance from shore, nutrient availability, temperature, salinity, and chlorophyll a, affected the distribution and co-occurrence of microbial groups. Our results highlight the crucial roles of environmental gradients in determining microbiome association and stability. These results improve our understanding of the survival strategies/adaptive mechanisms of microbial communities in response to environmental variation and provide new insights for protecting the ecosystems and maintaining the sustainability of ecological functions.

Accurate and Wide-Voltage-Range Modeling of Electrowetting with a Lattice Boltzmann Approach.

The lattice Boltzmann method (LBM) has been widely used in multi-phase fluid mechanics and is known to be more computationally efficient than the traditional method of numerically solving Navier-Stokes and Cahn-Hilliard equations. Electrowetting is an important component of interfacial sciences, in which the liquid-liquid and solid-liquid interfaces are tuned by electrostatics. Modeling electrowetting using the LBM can be categorized into surface and bulk methods. By modifying the surface tension scalar, the surface method easily reproduces the fundamental Young-Lippmann (YL) equation at low voltages but fails to capture contact angle saturation at high voltages. With fully coupled hydrodynamics and electrostatics in the form of spatially dependent matrices, the bulk method can successfully show contact angle saturation, but it is often unable to reproduce the YL equation due to its intrinsic inaccuracies. The inaccuracies are mainly due to the fact that while the hydrodynamics are all described by continuous physical quantities in the framework of diffusive interfaces, the interfacial electrostatics are governed by discontinuous electric fields caused by sheet charge density. In this paper, we show that accurately modeling electrowetting using the LBM is non-trivial. Additional modeling work, especially the treatment of interfacial electric fields, is needed to recover the fundamental YL equation at low voltages and predict contact angle saturation at high voltages, with a systematic model validation over key parameters and applications.

The Upconversion Luminescence of Ca3Sc2Si3O12:Yb3+,Er3+ and Its Application in Thermometry.

To develop novel luminescent materials for optical temperature measurement, a series of Yb3+- and Er3+-doped Ca3Sc2Si3O12 (CSS) upconversion (UC) phosphors were synthesized by the sol-gel combustion method. The crystal structure, phase purity, and element distribution of the samples were characterized by powder X-ray diffraction and a transmission electron microscope (TEM). The detailed study of the photoluminescence emission spectra of the samples shows that the addition of Yb3+ can greatly enhance the emission of Er3+ by effective energy transfer. The prepared Yb3+ and Er3+ co-doped CSS phosphors exhibit green emission bands near 522 and 555 nm and red emission bands near 658 nm, which correspond to the 2H11/2→4I15/2, 4S3/2→4I15/2, and 4F9/2→4I15/2 transitions of Er3+, respectively. The temperature-dependent behavior of the CSS:0.2Yb3+,0.02Er3+ sample was carefully studied by the fluorescence intensity ratio (FIR) technique. The results indicate the excellent sensitivity of the sample, with a maximum absolute sensitivity of 0.67% K-1 at 500 K and a relative sensitivity of 1.34% K-1 at 300 K. We demonstrate here that the temperature measurement performance of FIR technology using the CSS:Yb3+,Er3+ phosphor is not inferior to that of infrared thermal imaging thermometers. Therefore, CSS:Yb3+,Er3+ phosphors have great potential applications in the field of optical thermometry.

Collective Transport for Nonlinear Current-Voltage Characteristics of Doped Conducting Polymers.

Origin of nonlinear transport phenomena in conducting polymers has long been a topic of intense controversies. Most previous knowledge has attributed the macroscopic nonlinear I-V characteristics to individual behaviors of elementary resistors in the network. In this Letter, we show via a systematic dimensionality-dependent transport investigation, that understanding the nonlinear transport in conducting polymers must include the collective transport effect in a percolation network. The possible mediation of percolation threshold p_{c} by controlling the samples' dimensionality unveiled the collective effect in growth of percolation paths driven by electric field, enabling us to draw a smooth connection between two typically observed nonlinear phenomena, dissipative tunnelinglike and threshold-limited transport, which have been controversial for years. The possible microscopic origins of the collective transport are discussed within the Coulomb blockade theory.

Diazo probe-based chemical isotope labeling assisted liquid chromatography-tandem mass spectrometry analysis for sensitive determination of amino acids in biofluids.

Amino acids are important biomolecules and contribute to essential biological processes. Liquid chromatography tandem mass spectrometry (LC-MS) now is a powerful tool for the analysis of amino acid metabolites; however, the structural similarity and polarity of amino acids can lead to the poor chromatographic retention and low detection sensitivities. In this study, we used a pair of light and heavy isotopomers of diazo probes, d0/d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA/d5 -2-DMBA) to label amino acids. The paired MS probes of 2-DMBA and d5 -2-DMBA carry diazo groups that can efficiently and specifically react with the carboxyl group on free amino acid metabolites under mild conditions. Benefiting from the transfer of the 2-DMBA/d5 -2-DMBA to carboxyl group on amino acids, the ionization efficiencies of amino acids presented great enhancement during LC-MS analysis. The results suggested that the detection sensitivities of 17 amino acids increased by 9-133-fold upon 2-DMBA labeling, and the obtained limits of detection (LODs) of amino acids on-column ranged from 0.011 fmol-0.057 fmol. With the application of the developed method, we successfully achieved the sensitive and accurate detection of the 17 amino acids in microliter level of serum sample. Moreover, the contents of most amino acids were different in the serum from normal and B16F10-tumour mice, demonstrating that endogenous amino acids may play important roles in the regulation of tumors development. This developed method of chemical labeling of amino acids with diazo probes assisted LC-MS analysis provides a potentially valuable tool to investigate the relationships between amino acids metabolism and diseases.

Remodeling Tumor Immunogenicity with Dual-Activatable Binary CRISPR Nanomedicine for Cancer Immunotherapy.

The specific recognition of cancer cells by the body's immune system is an essential step in initiating antitumor immunity. However, the decreased expression of major histocompatibility complex class I (MHC-1) and overexpression of programmed death ligand 1 (PD-L1) causes insufficient tumor-associated antigens presentation and inactivation of T cells, which accounts for poor immunogenicity. To remodel tumor immunogenicity, herein, a dual-activatable binary CRISPR nanomedicine (DBCN) that can efficiently deliver a CRISPR system into tumor tissues and specifically control its activation is reported. This DBCN is made of a thioketal-cross-linked polyplex core and an acid-detachable polymer shell, which can maintain stability during blood circulation, while detaching a polymer shell to facilitate the cellular internalization of the CRISPR system after entering tumor tissues and ultimately activating gene editing under exogenous laser irradiation, thereby maximizing the therapeutic benefits and reducing potential safety concerns. With the collaborative application of multiple CRISPR systems, DBCN efficiently corrects both dysregulation of MHC-1 and PD-L1 expression in tumors, thus initiating robust T cell-dependent antitumor immune responses to inhibit malignant tumor growth, metastasis, and recurrence. Given the increasing abundance of CRISPR toolkits, this research provides an appealing therapeutic strategy and a universal delivery platform to develop more advanced CRISPR-based cancer treatments.

Comprehensive Profiling of Phosphomonoester Metabolites in Saccharomyces cerevisiae by the Chemical Isotope Labeling-LC-MS Method.

Phosphomonoesters are important biosynthetic and energy metabolism intermediates in microorganisms. A comprehensive analysis of phosphomonoester metabolites is of great significance for the understanding of their metabolic phosphorylation process and inner mechanism. In this study, we established a pair of isotope reagent d0/d5-2-diazomethyl-N-methyl-phenyl benzamide-labeling-based LC-MS method for the comprehensive analysis of phosphomonoester metabolites. By this method, the labeled phosphomonoester metabolites specifically produced characteristic isotope paired peaks with an m/z difference of 5.0314 in the MS1 spectra and a pair of diagnostic ions (m/z 320.0693/325.1077) in the MS2 spectra. Based on this, a diagnostic ion-based strategy was established for the rapid screening, identification, and relative quantification of phosphomonoester metabolites. Using this strategy, 42 phosphomonoester metabolites were highly accurately identified fromSaccharomyces cerevisiae (S. cerevisiae). Notably, two phosphomonoesters were first detected fromS. cerevisiae. The relative quantification results indicated that the contents of nine phosphomonoester metabolites including two intermediates (Ru5P and S7P) in the pentose phosphate pathway (PPP) were significantly different between lycopene-producible and wild-type S. cerevisiae. A further enzyme assay indicated that the activity of the PPP was closely related to the production of lycopene. Our findings provide new perspectives for the related mechanism study and valuable references for making informed microbial engineering decisions.

Ultrasensitive Determination of Sugar Phosphates in Trace Samples by Stable Isotope Chemical Labeling Combined with RPLC-MS.

Sugar phosphates are important metabolic intermediates in organisms and play a vital role in energy and central carbon metabolism. Profiling of sugar phosphates is of great significance but full of challenges due to their high structural similarity and low sensitivities in liquid chromatography (LC)-mass spectrometry (MS). In this study, we developed a novel stable isotope chemical labeling combined with the reversed-phase (RP)LC-MS method for ultrasensitive determination of sugar phosphates at the single-cell level. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, sugar phosphate isomers can obtain better separation and identification, and the detection sensitivities of sugar phosphates increased by 3.5-147 folds. The obtained limits of detection of sugar phosphates ranged from 5 to 16 pg/mL. Using this method, we achieved ultrasensitive and accurate quantification of 12 sugar phosphates in different trace biological samples. Benefiting from the improved separation and detection sensitivity, we successfully quantified five sugar phosphates (d-glucose 1-phosphate, d-mannose 6-phosphate, d-fructose 6-phosphate, d-glucose 6-phosphate, and seduheptulose 7-phosphate) in a single protoplast of Arabidopsis thaliana.

Combination of Plasma Electrolytic Processing and Mechanical Polishing for Single-Crystal 4H-SiC.

Single-crystal 4H-SiC is a typical third-generation semiconductor power-device material because of its excellent electronic and thermal properties. A novel polishing technique that combines plasma electrolytic processing and mechanical polishing (PEP-MP) was proposed in order to polish single-crystal 4H-SiC surfaces effectively. In the PEP-MP process, the single-crystal 4H-SiC surface is modified into a soft oxide layer, which is mainly made of SiO2 and a small amount of silicon oxycarbide by plasma electrolytic processing. Then, the modified oxide layer is easily removed by soft abrasives such as CeO2, whose hardness is much lower than that of single-crystal 4H-SiC. Finally a scratch-free and damage-free surface can be obtained. The hardness of the single-crystal 4H-SiC surface is greatly decreased from 2891.03 to 72.61 HV after plasma electrolytic processing. By scanning electron microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS) observation, the plasma electrolytic processing behaviors of single-crystal 4H-SiC are investigated. The scanning white light interferometer (SWLI) images of 4H-SiC surface processed by PEP-MP for 30 s shows that an ultra-smooth surface is obtained and the surface roughness decreased from Sz 607 nm, Ra 64.5 nm to Sz 60.1 nm, Ra 8.1 nm and the material removal rate (MRR) of PEP-MP is about 21.8 µm/h.

Photon-recycling effect in perovskites for photovoltaic applications: a Monte Carlo study.

Photon recycling has been shown to play an important role in the optoelectronic properties and device performance of perovskite solar cells recently. However, there lacks an analytical method to accurately predict the dynamics of charge carriers and photons and the device performance with photon recycling due to the complexity of multiple electron-photon conversion processes involved in photon recycling. We propose a model based on the Monte Carlo simulation method that combines charge carrier diffusion and photon radiation transport to analyze the effects of photon recycling on electron-photon dynamics and device performance of perovskite solar cells. We show that the carrier lifetime can be significantly boosted by photon recycling in the radiative limit, which yields a 37 meV increase in the open-circuit voltage for a 500 nm thick perovskite solar cell. Our results provide insights for the working mechanisms of perovskite solar cells, and the new model can be further applied to other types of solar cells with photon recycling.

Bubble Manipulation Driven by Alternating Current Electrowetting: Oscillation Modes and Surface Detachment.

In this paper, a millimeter-sized bubble in water pending on a substrate is manipulated by applying an alternating current (AC) electric field, known as electrowetting on dielectric. In this setup, standing waves on the bubble surface are observed. The amplitude of these waves varies with frequency, and three resonance peaks (21, 76, and 134 Hz) can be identified. By incorporating the nonlinear friction force for the contact line to an existing surface mode model, a significant improvement to explain the spectrum of the oscillations is obtained, predicting three peak positions, widths, and heights with good accuracy. We also show that bubble detachment correlates with the low-frequency resonance peak. It is found experimentally that if close enough to this peak, then bubbles at sufficiently high voltages are observed to detach from the substrate. This suggests that inertial effects can effectively promote bubble detachment. To confirm this hypothesis, the bubble dynamics is simulated with COMSOL using the full Navier-Stokes equation with a two-phase field and electrostatic stresses. It was found that the bubble experimental detachment process is quite well-reproduced in the simulation, confirming the role of fluid inertia for the detachment process. Given the nice correspondence between the experimental state diagrams and the theoretical modeling, this work contributes to identify a window for precise and reliable bubble manipulation by means of AC electrowetting.

Chemical Tagging Assisted Mass Spectrometry Analysis Enables Sensitive Determination of Phosphorylated Compounds in a Single Cell.

Polar phosphorylated metabolites are involved in a variety of biological processes and play vital roles in energetic metabolism, cofactor regeneration, and nucleic acid synthesis. However, it is often challenging to interrogate polar phosphorylated metabolites and compounds from biological samples. Liquid chromatography-mass spectrometry (LC/MS) now plays a central role in metabolomic studies. However, LC/MS-based approaches have been hampered by the issues of the low ionization efficiencies, low in vivo concentrations, and less chemical stability of polar phosphorylated metabolites. In this work, we synthesized paired reagents of light and heavy isotopomers, 2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (DMPI) and d3-(2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone (d3-DMPI). The paired reagents of DMPI and d3-DMPI carry diazo groups that can efficiently and selectively react with the phosphate group on polar phosphorylated metabolites under mild conditions. As a proof of concept, we found that the transfer of the indole heterocycle group from DMPI/d3-DMPI to ribonucleotides led to the significant increase of ionization efficiencies of ribonucleotides during LC/MS analysis. The detection sensitivities of these ribonucleotides increased by 25-1137-fold upon DMPI tagging with the limits of detection (LODs) being between 7 and 150 amol. With the developed method, we achieved the determination of all the 12 ribonucleotides from a single mammalian cell and from a single stamen of Arabidopsis thaliana. The method provides a valuable tool to investigate the dynamic changes of polar phosphorylated metabolites in a single cell under particular conditions.

Sensitive and Simultaneous Determination of Uridine Thiolation and Hydroxylation Modifications in Eukaryotic RNA by Derivatization Coupled with Mass Spectrometry Analysis.

The discovery of dynamic and reversible modifications in RNA expands their functional repertoires. Now, RNA modifications have been viewed as new regulators involved in a variety of biological processes. Among these modifications, thiolation is one kind of special modification in RNA. Several thiouridines have been identified to be present in RNA, and they are essential in the natural growth and metabolism of cells. However, detection of these thiouridines generally is challenging, and few studies could offer the quantitative levels of uridine modifications in RNA, which limits the in-depth elucidation of their functions. Herein, we developed a chemical derivatization in combination with mass spectrometry analysis for the sensitive and simultaneous determination of uridine thiolation and hydroxylation modifications in eukaryotic RNA. The chemical derivatization strategy enables the addition of easily ionizable groups to the uridine thiolation and hydroxylation modifications, leading up to a 339-fold increase in detection sensitivities of these modifications by mass spectrometry analysis. The limits of detection of these uridine modifications can be down to 17 amol. With the established method, we discovered and confirmed that a new modification of 5-hydroxyuridine (ho5U) was widely present in small RNAs of mammalian cells, expanding the diversity of RNA modifications. The developed method shows superior capability in determining low-abundance RNA modifications and may promote identifying new modifications in RNA, which should be valuable in uncovering the unknown functions of RNA modifications.