separation-immune-cell-subpopulations-peripheral-blood-samples-from

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

detection-polyfunctional-t-cells-children-vaccinated-with-japanese

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine <em>via</em> the Flow Cytometry Technique

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" ...

Capital Medical University, National Center for Children's Health

Chinese Academy of Medical Sciences

Capital Medical University, National Center for Children’s Health

2019RU016, Chinese Academy of Medical Sciences

Capital Medical University, China Rehabilitation Research Center

Protein glutamine methylation at GGQ sites of protein chain release factors plays a pivotal role in the termination of translation. We report here the crystal structure of the Escherichia coli HemK protein (N5)-glutamine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy). HemK contains two domains: a putative substrate binding domain at the N terminus consisting of a five helix bundle and a seven-stranded catalytic domain at the C terminus that harbors the binding site for AdoHcy. The two domains are linked by a beta-hairpin. Structure-guided sequence analysis of the HemK family revealed 11 invariant residues functioning in methyl-donor binding and catalysis of methyl transfer. The putative substrate-binding domains of HemK from E.coli and Thermotoga maritima are structurally similar, despite the fact that they share very little sequence similarity. When the two proteins are aligned structurally, the helical N-terminal domain is subject to approximately 10 degrees of hinge movement relative to the C-terminal domain. The apparent hinge mobility of the two domains may reflect functional importance during the reaction cycle. Comparative phylogenetic analysis of the hemK gene and its frequent neighbor gene, prfA, which encodes a major substrate, provides evidence for several examples of lateral gene transfer.

Structural characterization and comparative phylogenetic analysis of Escherichia coli HemK, a protein (N5)-glutamine methyltransferase.

Single-cell genomic analysis has grown rapidly in recent years and finds widespread applications in various fields of biology, including cancer biology, development, immunology, pre-implantation genetic diagnosis, and neurobiology. To date, the amplification bias, amplification uniformity and reproducibility of the three major single cell whole genome amplification methods (GenomePlex WGA4, MDA and MALBAC) have not been systematically investigated using mammalian cells. In this study, we amplified genomic DNA from individual hippocampal neurons using three single-cell DNA amplification methods, and sequenced them at shallow depth. We then systematically evaluated the GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. The MALBAC displays significant biases towards high GC content. We then attempted to correct the GC bias issue by developing a bioinformatics pipeline, which allows us to call CNVs in single cell sequencing data, and chromosome level and sub-chromosomal level CNVs among individual neurons can be detected. We also proposed a metric to determine the CNV detection limits. Overall, MALBAC and WGA4 have better performance than MDA in detecting CNVs. 

Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using hippocampal neurons.

Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic-resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (DO) metabolism is reported. Metabolic incorporation of DO into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% DO medium, the fastest among current technologies. Single-cell metabolism inactivation concentration (SC-MIC) is obtained in less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of DO metabolic incorporation and SC-MIC determination after 1 h antibiotic treatment and 30 min mixture of DO and antibiotics incubation of bacteria in urine or whole blood is demonstrated.

Rapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of DO Metabolic Incorporation in a Single Bacterium.

Plasmon-enhanced coherent Raman scattering microscopy has reached single-molecule detection sensitivity. Due to the different driven fields, there are significant differences between a coherent Raman scattering process and its plasmon-enhanced derivative. The commonly accepted line shapes for coherent anti-Stokes Raman scattering and stimulated Raman scattering do not hold for the plasmon-enhanced condition. Here, we present a theoretical model that describes the spectral line shapes in plasmon-enhanced coherent anti-Stokes Raman scattering (PECARS). Experimentally, we measured PECARS and plasmon-enhanced stimulated Raman scattering (PESRS) spectra of 4-mercaptopyridine adsorbed on the self-assembled Au nanoparticle (NP) substrate and aggregated Au NP colloids. The PECARS spectra show a nondispersive line shape, while the PESRS spectra exhibit a dispersive line shape. PECARS shows a higher signal to noise ratio and a larger enhancement factor than PESRS from the same specimen. It is verified that the nonresonant background in PECARS originates from the photoluminescence of nanostructures. The decoupling of background and the vibrational resonance component results in the nondispersive line shape in PECARS. More local electric field enhancements are involved in the PECARS process than in PESRS, which results in a higher enhancement factor in PECARS. The current work provides new insight into the mechanism of plasmon-enhanced coherent Raman scattering and helps to optimize the experimental design for ultrasensitive chemical imaging.

Plasmon-enhanced coherent anti-stokes Raman scattering vs plasmon-enhanced stimulated Raman scattering: Comparison of line shape and enhancement factor.

Ergosterol-targeting amphotericin B (AmB) is the first line of defense for life-threatening fungal infections. Two models have been proposed to illustrate AmB assembly in the cell membrane; one is the classical ion channel model in which AmB vertically forms transmembrane tunnel and the other is a recently proposed sterol sponge model where AmB is laterally adsorbed onto the membrane surface. To address this controversy, we use polarization-sensitive stimulated Raman scattering from fingerprint C═C stretching vibration to visualize AmB, ergosterol, and lipid in single fungal cells. Intracellular lipid droplet accumulation in response to AmB treatment is found. AmB is located in membrane and intracellular droplets. In the 16 strains studied, AmB residing inside cell membrane was highly ordered, and its orientation is primarily parallel to phospholipid acyl chains, supporting the ion channel model. Label-free imaging of AmB and chemical contents offers an analytical platform for developing low-toxicity, resistance-refractory antifungal agents.

Polarization-sensitive stimulated Raman scattering imaging resolves amphotericin B orientation in  membrane.

Mid-infrared photothermal microscopy is a new chemical imaging technology in which a visible beam senses the photothermal effect induced by a pulsed infrared laser. This technology provides infrared spectroscopic information at submicrometer spatial resolution and enables infrared spectroscopy and imaging of living cells and organisms. Yet, current mid-infrared photothermal imaging sensitivity suffers from a weak dependence of scattering on the temperature, and the image quality is vulnerable to the speckles caused by scattering. Here, we present a novel version of mid-infrared photothermal microscopy in which thermosensitive fluorescent probes are harnessed to sense the mid-infrared photothermal effect. The fluorescence intensity can be modulated at the level of 1% per Kelvin, which is 100 times larger than the modulation of scattering intensity. In addition, fluorescence emission is free of interference, thus much improving the image quality. Moreover, fluorophores can target specific organelles or biomolecules, thus augmenting the specificity of photothermal imaging. Spectral fidelity is confirmed through fingerprinting a single bacterium. Finally, the photobleaching issue is successfully addressed through the development of a wide-field fluorescence-detected mid-infrared photothermal microscope which allows video rate bond-selective imaging of biological specimens.

Fluorescence-Detected Mid-Infrared Photothermal Microscopy.

Spectroscopic stimulated Raman scattering (SRS) imaging has become a useful tool finding a broad range of applications. Yet, wider adoption is hindered by the bulky and environmentally sensitive solid-state optical parametric oscillator (OPO) in a current SRS microscope. Moreover, chemically informative multiwindow SRS imaging across C-H, C-D, and fingerprint Raman regions is challenging due to the slow wavelength tuning speed of the solid-state OPO. In this work, we present a multiwindow SRS imaging system based on a compact and robust fiber laser with rapid and wide tuning capability. To address the relative intensity noise intrinsic to a fiber laser, we implemented autobalanced detection, which enhances the signal-to-noise ratio of stimulated Raman loss imaging by 23 times. We demonstrate high-quality SRS metabolic imaging of fungi, cancer cells, and  across the C-H, C-D, and fingerprint Raman windows. Our results showcase the potential of the compact multiwindow SRS system for a broad range of applications.

Multiwindow SRS Imaging Using a Rapid Widely Tunable Fiber Laser.

Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.

Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering.

Stroke is the leading cause of death in China. However, recent data about the up-to-date stroke burden in China are limited.

Estimated Burden of Stroke in China in 2020.

To understand the varieties, evaluation, treatment, and prognosis of severe neurological diseases using the third NCU survey in China.

The development of neurocritical care in China from the perspective of evaluation and treatment of critical neurological diseases.

To explore the efficacy of a prediction model based on diffusion-weighted imaging (DWI) features extracted from deep learning (DL) and radiomics combined with clinical parameters and apparent diffusion coefficient (ADC) values to identify microsatellite instability (MSI) in endometrial cancer (EC).

ArticleTotal : 2	Year
Publication title Cited by 4	2022
Publication title Cited by 1	2022

Article	Year
Structural characterization and comparative phylogenetic analysis of Escherichia coli HemK, a protein (N5)-glutamine methyltransferase. Journal of molecular biology\| PubMed ID: 15223314	2004
Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using hippocampal neurons. Scientific reports\| PubMed ID: 26091148	2015
Rapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of DO Metabolic Incorporation in a Single Bacterium. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 33042757	2020
Plasmon-enhanced coherent anti-stokes Raman scattering vs plasmon-enhanced stimulated Raman scattering: Comparison of line shape and enhancement factor. The Journal of chemical physics\| PubMed ID: 33499625	2021
Polarization-sensitive stimulated Raman scattering imaging resolves amphotericin B orientation in membrane. Science advances\| PubMed ID: 33523971	2021
Fluorescence-Detected Mid-Infrared Photothermal Microscopy. Journal of the American Chemical Society\| PubMed ID: 34264654	2021
Multiwindow SRS Imaging Using a Rapid Widely Tunable Fiber Laser. Analytical chemistry\| PubMed ID: 34787995	2021
Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering. Nature communications\| PubMed ID: 34876556	2021
Estimated Burden of Stroke in China in 2020. JAMA network open\| PubMed ID: 36862407	2023
The development of neurocritical care in China from the perspective of evaluation and treatment of critical neurological diseases. Frontiers in neurology\| PubMed ID: 36895910	2023
A prediction model based on deep learning and radiomics features of DWI for the assessment of microsatellite instability in endometrial cancer. Cancer medicine\| PubMed ID: 39171859	2024

Meng Zhang

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