基于正交质谱的N-糖组谱揭示哈夫病潜在病原学

食用煮熟的小龙虾可能导致哈夫病(HD),该病被认为是由不明毒素引起的,而病因尚不清楚。N-糖组的剖析将促进破译疾病的分子机制,而HD相关的糖基化从未被探索过。2019-2020年期间,本研究团队招募了来自武汉市疾病预防控制中心的90份HD患者和对照组血清样本。本文中,采用基于高通量的正交质谱对HD中血清和血清衍生的IgG的N-糖组谱进行了表征。数据显示,HD与总血清糖蛋白的核心岩藻糖基化和单半乳糖醇化升高有关。血清IgG水平是HD患者的良好指标。此外,IgG的差异半乳糖基化和唾液酸化与HD密切相关。值得注意的是,IgG1和IgG2的半乳糖基化和唾液酸化的变化具有亚类特异性。有意义的是,IgG2或IgG3/4的唾液酸化和半乳糖基化改变与HD的临床标志物密切相关。本研究表明差异化IgG N-糖基化与HD的关联,为这种罕见疾病的病因提供了新的见解。

Microfluidics-based strategies for molecular diagnostics of infectious diseases

Traditional diagnostic strategies for infectious disease detection require benchtop instruments that are inappropriate for point-of-care testing(POCT). Emerging microfluidics, a highly miniaturized, automatic, and integrated technology,are a potential substitute for traditional methods in performing rapid, low-cost, accurate, and on-site diagnoses.Molecular diagnostics are widely used in microfluidic devices as the most effective approaches for pathogen detection.This review summarizes the latest advances in microfluidics-based molecular diagnostics for infectious diseases from academic perspectives and industrial outlooks. First, we introduce the typical on-chip nucleic acid processes,including sample preprocessing, amplification, and signal read-out. Then, four categories of microfluidic platforms are compared with respect to features, merits, and demerits. We further discuss application of the digital assay in absolute nucleic acid quantification. Both the classic and recent microfluidics-based commercial molecular diagnostic devices are summarized as proof of the current market status. Finally, we propose future directions for microfluidics-based infectious disease diagnosis.

Candidate reference measurement procedure for serum 17-hydroxyprogesterone quantification via isotope dilution liquid chromatography–tandem mass spectrometry

Background:Accurate quantification of 17-hydroxyprogesterone(17-OHP)in serum is vital for clinical and research applications.However,inter-laboratory variability in test results exists owing to the lack of a standardized reference measurement procedure(RMP).Therefore,in this study,we developed a highly accurate,cost-effective,and user-friendly candidate RMP(cRMP)for analyzing 17-OHP in serum.Methods:We quantified 17-OHP in serum using a one-step liquid–liquid extraction method with the addition of 17-OHP-^(13)C_(3),followed by liquid chromatography–tandem mass spectrometry.The ability of these methods to suppress interference was evaluated by chromatographic analysis.We assessed accuracy,specificity,the lower limit of quantitation,linearity,and matrix effects by following the standards specified by the Clinical and Laboratory Standards Institute.The consistency between the developed cRMP and the chemiluminescence method was evaluated through experiments with 120 clinical samples.Results:The developed cRMP required only 8 min for accurate quantification of serum 17-OHP without bias from matrix effects or interference from 19 metabolites added as potential interferents.The method exhibited favorable measurement performance,with a quantitation limit of 0.086 ng/mL,linear range of 0.1–400 ng/mL,a total imprecision of≤2.90%,spike recovery of 100.1%–100.6%,and relative deviations from assigned target values(RfB Institution)of−2.91%to 1.10%.The cRMP demonstrated good consistency with the conventional assay(chemiluminescence method),with a correlation coefficient R of 0.96977.Conclusion:A cRMP with high accuracy,cost-effectiveness,and convenient operation was developed for quantifying 17-OHP in serum.Its simplicity and robust performance make it an invaluable addition to the arsenal of analytical tools available for laboratories.This method can enhance the accuracy and reliability of 17-OHP measurements across various laboratories.

Facile synthesis of suvorexant,an orexin receptor antagonist,via a chiral diazepane intermediate

A facile synthesis of suvorexant, an orexin receptor antagonist, is described. The key intermediate 6 was prepared from R-3-aminobutyric acid through protection, condensation, deprotection, cyclization, and hydrogenation steps. The title product was obtained with a total yield of 31% （〉99% ee） after eight linear steps using commercially available raw materials.

Modulation of tumor microenvironment by metal-organic-framework-derived nanoenzyme for enhancing nucleus-targeted photodynamic therapy

Photodynamic therapy(PDT)is a promising strategy for tumor treatment.Still,its therapeutic efficacy is compromised by the unsatisfactory cytotoxicity to specific subcellular organelles and insidious tumor microenvironment properties like hypoxia and high glutathione levels.Here,we fabricated a novel nanoenzyme that derived from metal-organic framework(MOF)with intrinsic catalase-like activities to decompose H2O2 to O2 and simultaneous glutathione consumption for enhancing PDT efficacy.The obtained Mn3O4 nanoparticle shows a larger pore size and surface area compared to native MOF particles,which can be used to load high dose photosensitizer.When decorated with AS1411 aptamer and polyethylene glycol(PEG),the obtained Mn3O4-PEG@C&A particle exhibits excellent stability and cell nucleus targeting ability.Remarkably,Mn3O4-PEG@C&A particle inhibited the tumor growth in the mouse model with high efficacy without any biotoxicity.This is the first report that applied MOF-derived nanoparticle to nucleus-targeted PDT.It may provide a new approach for designing functional nanoenzyme to subcellular organelles-targeted tumor modulation.

SARS-CoV-2-specific immune response in COVID-19 convalescent individuals

We collected blood from coronavirus disease 2019(COVID-19)convalescent individuals and investigated SARS-CoV-2-specific humoral and cellular immunity in these discharged patients.Follow-up analysis in a cohort of 171 patients at 4-11 months after the onset revealed high levels of IgG antibodies.A total of 78.1%(164/210)of the specimens tested positive for neutralizing antibody(NAb).SARS-CoV-2 antigen peptide pools-stimulated-IL-2 and-IFN-y response can distinguish COVID-19 convalescent individuals from healthy donors.Interestingly,NAb survival was significantly affected by the antigen peptide pools-stimulated-IL-2 response,-IL-8 response,and-IFN-y response.The antigen peptide pools-activated CD8+T cell counts were correlated with NAb.The antigen peptide pools-activated natural killer(NK)cell counts in convalescent individuals were correlated with NAb and disease severity.Our data suggested that the development of NAb is associated with the activation of T cells and NK cells.Our work provides a basis for further analysis of the protective immunity to SARS-CoV-2 and for understanding the pathogenesis of COVID-19.It also has implications for the development of an effective vaccine for SARS-CoV-2 infection.

A time-coded multi-concentration microfluidic chemical waveform generator for high-throughput probing suspension single-cell signaling

The cellular response to the complex extracellular microenvironment is highly dynamic in time and type of extracellular matrix.Accurately reconstructing this process and analyzing the changes in receptor conformation on the cell membrane surface and intracellular or intercellular signaling has been a major challenge in analytical chemistry and biophysical methodology.In this paper,a time-coded multiconcentration microfluidic chemical waveform generator was developed for the dynamic signaling probing with single-cell array of high temporal resolution,high throughput,and multi-concentration combination stimulation.Based on innovative microchannel structure,sophisticated external control methods and multiplexing technology,the system not only allowed for temporally sequential permutations of the four concentrations of stimuli(time code),but also generated pulsed and continuous waveforms at different frequencies in a highly controllable manner.Furthermore,the single-cell trap array was set up to efficiently capture cells in suspension,dramatically increasing throughput and reducing experiment preparation time.The maximum frequency of the platform was 1 Hz,and one cell could be stimulated at multiple frequencies.To show the ability of the system to investigate rapid biochemical events in high throughput,pulse stimulation and continuous stimulation of different frequencies and different time codes,combined with four concentrations of histamine(HA),were generated for probing G protein-coupled receptor(GPCR)signaling in He La cells.Then,statistical analysis was performed for the mean peak height and mean peak area of the cellular response.We believe that the time-coded multi-concentration microfluidic chemical waveform generator will provide a novel strategy for analytical chemistry,biophysics,cell signaling,and individualized medicine applications.