Discovery of Novel ERα and Aromatase Dual-Targeting PROTAC Degraders to Overcome Endocrine-Resistant Breast Cancer.

Estrogen receptor α (ERα) plays a pivotal role in the proliferation, differentiation, and migration of breast cancer (BC) cells, and aromatase (ARO) is a crucial enzyme in estrogen synthesis. Hence, it is necessary to inhibit estrogen production or the activity of ERα for the treatment of estrogen receptor-positive (ER+) BC. Herein, we present a new category of dual-targeting PROTAC degraders designed to specifically target ERα and ARO. Among them, compound 18c bifunctionally degrades and inhibits ERα/ARO, thus effectively suppressing the proliferation of MCF-7 cells while showing negligible cytotoxicity to normal cells. In vivo, 18c promotes the degradation of ERα and ARO and inhibits the growth of MCF-7 xenograft tumors. Finally, compound 18c demonstrates promising antiproliferative and ERα degradation activity against the ERαMUT cells. These findings suggest that 18c, being the inaugural dual-targeting degrader for ERα and ARO, warrants further advancement for the management of BC and the surmounting of endocrine resistance.

Targeting METTL3 reprograms the tumor microenvironment to improve cancer immunotherapy.

The tumor microenvironment (TME) is a heterogeneous ecosystem containing cancer cells, immune cells, stromal cells, cytokines, and chemokines which together govern tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), a core catalytic subunit for RNA N6-methyladenosine (m6A) modification, plays a crucial role in regulating various physiological and pathological processes. Whether and how METTL3 regulates the TME and anti-tumor immunity in non-small-cell lung cancer (NSCLC) remain poorly understood. Here, we report that METTL3 elevates expression of pro-tumorigenic chemokines including CXCL1, CXCL5, and CCL20, and destabilizes PD-L1 mRNA in an m6A-dependent manner, thereby shaping a non-inflamed TME. Thus, inhibiting METTL3 reprograms a more inflamed TME that renders anti-PD-1 therapy more effective in several murine lung tumor models. Clinically, NSCLC patients who exhibit low-METTL3 expression have a better prognosis when receiving anti-PD-1 therapy. Collectively, our study highlights targeting METTL3 as a promising strategy to improve immunotherapy in NSCLC patients.

Simultaneous Quantification of Seven Antifungal Agents in Human Serum Using Liquid Chromatography-Tandem Mass Spectrometry.

Systemic antifungal agents are essential for high-risk patients undergoing immunosuppressive therapy or cancer chemotherapy because of the rapid increase in opportunistic fungal infections. Therapeutic drug monitoring is crucial to ensuring the efficacy and safety of antifungal agents owing to their pharmacokinetic variability. In the present study, we developed and validated a quantitative method for the simultaneous detection of seven commonly used antifungal drugs (amphotericin B, isavuconazole, voriconazole, fluconazole, posaconazole, caspofungin, and micafungin) using liquid chromatography-tandem mass spectrometry. Methanol (containing 0.1% formic acid) was used for protein precipitation and only 50 µL of serum was required for the analysis. Chromatographic separation was conducted using a Waters Acquity UPLC C8 column, and one stable isotope-labeled agent and two analogs were used as internal standards. The calibration curves ranged from 0.1 to 50 µg/mL for all agents, and the correlation coefficient (R2) for all calibration curves was above 0.9835. The intra-day precision (1.2-11.2%), inter-day precision (2.4-13.2%), and mean bias values (-10.9 to 13.6%) were within an acceptable range of ±15%. Successful implementation of the developed method in clinical practice would facilitate the effective monitoring of these antifungal agents.

A mitochondrial EglN1-AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress.

Mitochondria play essential roles in cancer cell adaptation to hypoxia, but the underlying mechanisms remain elusive. Through mitochondrial proteomic profiling, we here find that the prolyl hydroxylase EglN1 (PHD2) accumulates on mitochondria under hypoxia. EglN1 substrate-binding region in the ß2ß3 loop is responsible for its mitochondrial translocation and contributes to breast tumor growth. Furthermore, we identify AMP-activated protein kinase alpha (AMPKα) as an EglN1 substrate on mitochondria. The EglN1-AMPKα interaction is essential for their mutual mitochondrial translocation. After EglN1 prolyl-hydroxylates AMPKα under normoxia, they rapidly dissociate following prolyl-hydroxylation, leading to their immediate release from mitochondria. In contrast, hypoxia results in constant EglN1-AMPKα interaction and their accumulation on mitochondria, leading to the formation of a Ca2+ /calmodulin-dependent protein kinase 2 (CaMKK2)-EglN1-AMPKα complex to activate AMPKα phosphorylation, ensuring metabolic homeostasis and breast tumor growth. Our findings identify EglN1 as an oxygen-sensitive metabolic checkpoint signaling hypoxic stress to mitochondria through its ß2ß3 loop region, suggesting a potential therapeutic target for breast cancer.

Glucose-responsive, antioxidative HA-PBA-FA/EN106 hydrogel enhanced diabetic wound healing through modulation of FEM1b-FNIP1 axis and promoting angiogenesis.

The diabetic wounds remain to be unsettled clinically, with chronic wounds characterized by drug-resistant bacterial infections, compromised angiogenesis and oxidative damage to the microenvironment. To ameliorate oxidative stress and applying antioxidant treatment in the wound site, we explore the function of folliculin-interacting protein 1 (FNIP1), a mitochondrial gatekeeper protein works to alter mitochondrial morphology, reduce oxidative phosphorylation and protect cells from unwarranted ROS accumulation. And our in vitro experiments showed the effects of FNIP1 in ameliorating oxidative stress and rescued impaired angiogenesis of HUVECs in high glucose environment. To realize the drug delivery and local regulation of FNIP1 in diabetic wound sites, a novel designed glucose-responsive HA-PBA-FA/EN106 hydrogel is introduced for improving diabetic wound healing. Due to the dynamic phenylboronate ester structure with a phenylboronic acid group between hyaluronic acid (HA) and phenylboronic acid (PBA), the hydrogel is able to realize a glucose-responsive release of drugs. Fulvic acid (FA) is added in the hydrogel, which not only severs as crosslinking agent but also provides antibacterial and anti-inflammatory abilities. Moreover, the release of FEM1b-FNIP1 axis inhibitor EN106 ameliorated oxidative stress and stimulated angiogenesis through FEM1b-FNIP1 axis regulation. These in vivo and in vitro results demonstrated that accelerated diabetic wounds repair with the use of the HA-PBA-FA/EN106 hydrogel, which may provide a promising strategy for chronic diabetic wound repair.

Aptamer-based sample purification for mass spectrometric quantification of trastuzumab in human serum.

In this study, we developed a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify trastuzumab in human serum using aptamers for sample purification. Trastuzumab was extracted from serum samples using the capture probe based on its aptamer CH1S-3, followed by reduction, alkylation, trypsin digestion, and quantification using LC-MS/MS. Additionally, a unique peptide, FTISADTSK, was employed as a surrogate peptide and quantified, and *FTISADTSK (13C915N-labeled phenylalanine) was used as an internal standard to minimize variability in detection among the samples. The detection range for this method was 0.5-250 µg/mL, with a high correlation coefficient (r2 > 0.99). The intra- and inter-day precision (%CV, the coefficient of variation) of the quality control samples was less than 12.7%, and the accuracy (%bias) was below 8.64%. After optimization and verification, this assay was used to determine trastuzumab levels in clinical human serum samples. The results indicated that the trastuzumab concentrations had an approximate 4-fold difference among ten patients (range: 11.80-41.90 µg/mL). This study provides a novel approach for the accurate and quantitative monitoring of the mAb-trastuzumab.

Identification of molecular subtypes based on liquid-liquid phase separation and cross-talk with immunological phenotype in bladder cancer.

Background: Mounting evidence has demonstrated that an imbalance in liquid-liquid phase separation (LLPS) can induce alteration in the spatiotemporal coordination of biomolecular condensates, which plays a role in carcinogenesis and cachexia. However, the role of LLPS in the occurrence and progression of bladder cancer (BLCA) remains to be elucidated. Identifying the role of LLPS in carcinogenesis may aid in cancer therapeutics. Methods: A total of 1,351 BLCA samples from six cohorts were retrieved from publicly available databases like The Cancer Genome Atlas, Gene Expression Omnibus, and ArrayExpress. The samples were divided into three distinct clusters, and their multi-dimensional heterogeneities were explored. The LLPS patterns of all patients were determined based on the LLPS-related risk score (LLPSRS), and its multifaceted landscape was depicted and experimentally validated at the multi-omics level. Finally, a cytotoxicity-related and LLPSRS-based classifier was established to predict the patient's response to immune checkpoint blockade (ICB) treatment. Results: Three LLPS-related subtypes were identified and validated. The differences in prognosis, tumor microenvironment (TME) features, cancer hallmarks, and certain signatures of the three LLPS-related subtypes were validated. LLPSRS was calculated, which could be used as a prognostic biomarker. A close correlation was observed between clinicopathological features, genomic variations, biological mechanisms, immune infiltration in TME, chemosensitivity, and LLPSRS. Furthermore, our classifier could effectively predict immunotherapy response in patients with BLCA. Conclusions: Our study identified a novel categorization of BLCA patients based on LLPS. The LLPSRS could predict the prognosis of patients and aid in designing personalized medicine. Further, our binary classifier could effectively predict patients' sensitivity to immunotherapy.

Comparative metabolic profiling of wild Cordyceps species and their substituents by liquid chromatography-tandem mass spectrometry.

Cordyceps is a genus of ascomycete fungi and used widely in fungal drugs. However, in-depth studies of the metabolites of wild Cordyceps species and their substituents are lacking. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics analysis was carried out to comprehensively profile the metabolites in wild Chinese Cordyceps species (Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora 2007) from Naqu (NCs) and Yushu (YCs) and their substituents including artificially cultivated Cordyceps species (CCs) and mycelia. A total of 901 metabolites were identified in these samples, including lipids, amino acids, nucleosides, carbohydrates, organic acids, coenzymes, vitamins, alkaloids and their derivatives. Univariate and multivariate statistical analyses revealed remarkable differences and significantly different metabolites among them. Seventy amino acid-relevant metabolites were analyzed quantitatively in four samples for the first time. The four samples contained abundant L-glutamic acid and oxidized glutathione as well as multiple unique amino acid-relevant metabolites (e.g., 3-chloro-L-tyrosine, 6-aminocaproic acid, L-theanine, anserine, γ-glutamyl-cysteine). Collectively, our study provides rich metabolic information of wild Cordyceps species and their substituents, which could facilitate their quality control and optimal utilization.

Efficacy and safety of adjunctive antiseizure medications for dravet syndrome: A systematic review and network meta-analysis.

Purpose: Recently, the U.S. Food and Drug Administration (FDA) approved stiripentol, cannabidiol, and fenfluramine to treat patients with Dravet syndrome (DS). Moreover, soticlestat was determined as a promising new drug for the treatment of DS as it has good efficacy and safety. However, the efficacy and safety of these drugs have not yet been evaluated in "head-to-head" trials. This study aimed to compare and evaluate the efficacy and safety of these adjunctive antiseizure medications in the treatment of DS. Methods: We searched in PubMed, Embase, Cochrane Library, and Web of Science databases for randomized controlled trials (RCTs) and open-label extension (OLE) studies in patients with DS. We performed a random-effect meta-analysis of OLE studies and a network meta-analysis for RCTs to evaluate the efficacy and safety of antiseizure medications in the treatment of DS. Primary efficacy outcomes were defined as a ≥50% reduction in seizure frequency compared with baseline. Furthermore, safety evaluation indicators were defined as the incidence of adverse events (AEs) and serious adverse events (SAEs) during treatment. Relative ranking was assessed using the surface under the cumulative ranking curve (SUCRA) probabilities. Results: Seven RCTs involving four antiseizure medications (stiripentol, cannabidiol, fenfluramine, and soticlestat) and a total of 634 patients were included in the analysis. According to the SUCRA results, all four drugs significantly reduced the frequency of seizures compared with the placebo. Soticlestat was the most likely to reduce seizure frequency by ≥50% compared to the baseline [risk ratio (RR): 19.32; 95% confidence interval (CI): 1.20-311.40], followed by stiripentol and fenfluramine. Stiripentol was ranked highest for the near percentage reduction in the seizure rate from baseline [RR: 12.33; 95% CI: 1.71-89.17] and the occurrence of any treatment-emergent adverse events [RR: 3.73; 95% CI: 1.65-8.43] and serious adverse events [RR: 4.76; 95% CI: 0.61-37.28]. A total of ten OLE studies containing 1,121 patients were included in our study. According to the results of the meta-analysis, the order of probability of reducing seizure frequency by ≥50% was fenfluramine (0.715, 95% CI: 0.621-0.808), stiripentol (0.604, 95% CI: 0.502-0.706), cannabidiol (0.448, 95% CI: 0.403-0.493). And the probability of occurrence of AEs is ranked as fenfluramine(0.832, 95% CI: 0.795-0.869), cannabidiol (0.825, 95% CI:0.701-0.950), stiripentol (0.823, 95% CI: 0.707-0.938), soticlestat (0.688, 95% CI: 0.413-0.890). Conclusion: According to the results of indirect comparison of efficacy and safety, cannabidiol is slightly inferior to the other three antiseizure medications in terms of efficacy and safety. Soticlestat, fenfluramine, and stripentol may have little difference in efficacy, but soticlestat and fenfluramine are safer. Soticlestat is probably the best adjunctive antiseizure medication, followed by fenfluramine. This conclusion is consistent with the comparison of long-term efficacy and safety.

Using Haemocoagulase Agkistrodon in Patients Undergoing Transurethral Plasmakinetic Resection of the Prostate: A Pilot, Real-World, and Propensity Score-Matched Study.

Objectives: To compare the clinical outcomes of using different hemostatic agents after transurethral plasmakinetic resection of the prostate (TUPKP) in benign prostatic hyperplasia (BPH) patients. Methods: The patients were divided into 5 groups according to the hemostatic agents used after TUPKP, including the haemocoagulase agkistrodon for injection (HCA), hemocoagulase for injection (HC), hemocoagulase bothrops atrox for injection (HCB), ethylenediamine diaceturate injection (EDD), and tranexamic acid (TXA). Propensity score matching was performed based on age, body mass index, prostate volume, hypertension status, fasting blood glucose, smoking, and drinking history. The hospitalization time, bladder irrigation time, indwelling catheterization time, the patency of urine flow, and blood transfusion records were used as outcome indicators to compare the clinical effects of these five agents. Results: We finally matched 65 pairs receiving HCA or HC, 71 pairs receiving HCA or HCB, 38 pairs receiving HCA or TXA, and 29 pairs receiving HCA or EDD. Compared with HC, HCA given during the perioperative period significantly reduced the median hospitalization time [7.00 days (5.00, 8.00) vs. 9.00 days (8.00, 10.00); p < 0.001] and median catheterization time (109.00 hours [88.00, 129.00] vs. 164.00 hours [114.00, 189.00], p < 0.001). Compared with EDD, the median hospitalization time (7.00 days [6.00, 8.00] vs. 10.00 days [8.00, 11.00]; p < 0.001) and median catheterization time (113.00 hours [95.00, 143.00] vs. 160.00 hours [139.00, 168.00]; p < 0.001) were also significant shorter in HCA group. Compared with HCB, median bladder irrigation time (45.00 hours [27.00, 71.00] vs. 49.00 hours [45.00, 72.00]; p = 0.04) was shorter in the HCA group. However, there were no statistical differences in outcomes between HCA and TXA. Conclusions: HCA probably has an advantage over HC, HCB, and EDD in reducing the hospitalization time, catheterization time, and bladder irrigation time among BPH patients undergoing TUPKP.

(±)-Walskiiglucinol A, a pair of rearranged acylphloroglucinol derivative enantiomers from Hypericum przewalskii.

(±)-Walskiiglucinol A (1a/1b), a pair of rearranged acylphloroglucinol derivatives with a new carbon skeleton, was obtained from Hypericum przewalskii. Compounds 1a/1b were the first examples of naturally occurring acylphloroglucinol derivatives possessing a unique 1-oxaspiro[4.4]nonane core bearing a new 5/5 ring system. Their planar and relative structures were identified by extensive spectroscopic analysis and NMR chemical shift calculations with DP4+ probability analysis, and their absolute configurations were determined by electronic circular dichroism (ECD) calculations. A plausible biogenetic pathway of 1a/1b was proposed in which the breakage of the C-2/C-3 linkage via a retro-Claisen reaction and the cyclization between C-3 and C-1 were proposed as key steps. The isolates were evaluated for cytotoxic activities against a panel of cancer cell lines and anti-inflammatory activities against lipopolysaccharide (LPS)-induced NO production, and compounds 1a/1b showed moderate cytotoxic activities with IC50 values ranging from 9.72 to 36.75 µM.

Global and Regional Burden of Bacterial Antimicrobial Resistance in Urinary Tract Infections in 2019.

BACKGROUND: There are still no detailed data about the burden of bacterial antimicrobial resistance (AMR) in urinary tract infections (UTI). Concrete knowledge of global and regional bacterial AMR data is crucial for developing informed programs and policies to control bacterial AMR and for prudent use of antibiotics to optimize antibiotic therapy in patients with UTI. This study aimed to provide comprehensive global and regional estimates for the AMR burden of UTI in 2019. METHODS: Data were obtained from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD), including death, disability-adjusted life-years (DALYs), year lived with disability (YLD), and years of life lost (YLL) for bacterial AMR in UTI for 7 GBD super-regions, 21 regions, 14 pathogens, 13 antibiotic classes, and 66 pathogen-antibiotic combinations in 2019. The estimates were based on two counterfactual scenarios: drug-susceptible infection and no infection. RESULTS: Globally, there were 64.89 thousand deaths (95% uncertainty interval [UI]: 45.86-93.35) attributed to and 0.26 million deaths (95% UI: 0.18-0.36) associated with bacterial AMR in UTI in 2019. Among regions, the all-age death rates were higher in southern Latin America, tropical Latin America, and Europe and lower in sub-Saharan Africa. Escherichia coli and Klebsiella pneumoniae accounted for more than 50% of deaths attributable to and associated with AMR, and resistance was high among multiple types of antibiotic class, including fluoroquinolones, carbapenems, and third-generation cephalosporins. There were 2 pathogen-drug combinations that caused more than 6000 resistance-attributable deaths: third-generation cephalosporin-resistant Escherichia coli and fluoroquinolone-resistant Escherichia coli. CONCLUSIONS: AMR in UTI is an unignorable health problem, both for the management of urology disease and for global antibiotic resistance. Special tailored strategies, including enhanced surveillance and rational use of antibiotics, should be developed for different regions according to the region-specific pathogen-antibiotic situations and resources.

A density-tunable liquid-phase microextraction system based on deep eutectic solvents for the determination of polycyclic aromatic hydrocarbons in tea, medicinal herbs and liquid foods.

A novel density-tunable liquid-phase microextraction (DT-LPME) system was developed with high-density deep eutectic solvents (DESs) as extractant and low-density organic solvents as emulsifier and density regulator. DES-rich phase was induced to form in the bottom or in the top by adjusting the emulsifier amount. This system was used to directly extract polycyclic aromatic hydrocarbons (PAHs) from liquid and solid foods, and the obtained DES-rich phase was easy to be collected for quantification. The method (LPME with HPLC-fluorescence detector) has linearity (R2 > 0.9974), detection limits of 0.6-4.2 ng L-1 for liquid foods and 0.05-0.35 ng g-1 for solid foods, recoveries of 86.2-114.9%, and intra-day/inter-day RSDs below 6.6%. The method was applied to detect PAHs in real samples, and the PAHs residue was found in honey and five solid foods. The DT-LPME method is simple, fast, green and suitable for direct extraction of analytes from both liquid and solid samples.

A green liquid chromatography method for rapid determination of ergosterol in edible fungi based on matrix solid-phase dispersion extraction and a core-shell column.

Developing a green analytical method for the analysis of components in food samples is an important research aspect of liquid chromatography (LC). The traditional LC method usually consumes a lot of toxic solvent for sample extraction and LC separation. In the current study, a green analytical method for the rapid determination of ergosterol in edible fungi was established. The sample was extracted and purified by matrix solid-phase dispersion (MSPD) with a green solution (ethanol and water). The LC separation was performed using a Poroshell 120 SB-C18 (4.6 × 30 mm, 2.7 µm) column with a green mobile phase (94% ethanol) at a flow rate of 1.0 mL min-1. The detection wavelength was set at 283 nm. The calibration curve of ergosterol showed good linearity (R = 0.9999) within the test range (4.21-25.27 µg mL-1). The RSD of precision was less than 2.0% and the recovery was 100.4% (RSD = 3.23%). The developed method was successfully applied to quantitative analysis of ergosterol in six edible fungi and the contents of ergosterol were in the range of 1.68-4.02 mg g-1. Only 11.5 mL ethanol water solution was used in the sample extraction and LC separation in the newly developed method, and no toxic organic solvents were used. The total analysis time was less than 15.5 min, about 12-14 min for sample extraction and 1.5 min for LC analysis. This method was environmentally friendly and time-saving, which is helpful to improve the quality evaluation of edible fungi.

A DNA Methylation-Based Panel for the Prognosis and Dagnosis of Patients With Breast Cancer and Its Mechanisms.

OBJECTIVE: To identify DNA methylation related biomarkers in patients with breast cancer (BC). MATERIALS AND METHODS: A total of seven BC methylation studies including 1,438 BC patients or breast tissues were included in this study. An elastic net regularized Cox proportional hazards regression (CPH) model was used to build a multi-5'-C-phosphate-G-3' methylation panel. The diagnosis and prognosis power of the panel was evaluated and validated using a Kaplan-Meier curve, univariate and multivariable CPH, subgroup analysis. A nomogram containing the panel was developed. The relationships between the panel-based methylation risk and the immune landscape and genomic metrics were investigated. RESULTS: Sixty-eight CpG sites were significantly correlated with the overall survival (OS) of BC patients, and based on the result of penalized CPH, a 28-CpG site based multi CpG methylation panel was found. The prognosis and diagnosis role of the panel was validated in the discovery set, validation set, and six independent cohorts, which indicated that higher methylation risk was associated with poor OS, and the panel outperformed currently available biomarkers and remained an independent factor after adjusting for other clinical features. The methylation risk was negatively correlated with innated and adaptive immune cells, and positively correlated with total mutation load, SCNA, and MATH. CONCLUSIONS: We validated a multi CpG methylation panel that could independently predict the OS of BC patients. The Th2-mediated tumor promotion effect-suppression of innate and adaptive immunity-participated in the progression of high-risk BC. Patients with high methylation risk were associated with tumor heterogeneity and poor survival.

Fast quantification of free amino acids in food by microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry.

A method based on microfluidic voltage-assisted liquid desorption electrospray ionization-tandem mass spectrometry (VAL-DESI-MS/MS) has been developed for fast quantification of free amino acids in food. Food extracts were transferred to the microfluidic platform and analyzed by liquid desorption ESI-MS/MS. Deuterated aspartic acid (i.e., 2,2,3-d3-Asp) was used as internal standard for analysis. The method had linear calibration curves with r2 values > 0.998. Limits of detection were at the level of sub µM for the amino acids tested, i.e., glutamic acid (Glu), arginine (Arg), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). To validate the proposed method in food analysis, extracts of Cordyceps fungi were analyzed. Amino acid contents were found in the range from 0.63 mg/g (Tyr in Cordyceps sinensis) to 4.44 mg/g (Glu in Cordyceps militaris). Assay repeatability (RSD) was ≤ 5.2% for all the five amino acids measured in all the samples analyzed. Recovery was found in the range from 95.8 to 105.1% at two spiking concentrations of 0.250 mg/g and 1.00 mg/g. These results prove that the proposed microfluidic VAL-DESI-MS/MS method offers a quick and convenient means of quantifying free amino acids with accuracy and repeatability. Therefore, it may have potential in food analysis for nutritional and quality assessment purposes. Graphical abstract.

Simultaneous Quantification of Adenosine and Deoxyadenosine Isomers in Foods with High Sensitivity.

Simultaneous quantification of adenosine and deoxyadenosine isomers, including 2'-deoxyadenosine (dA) and 3'-deoxyadenosine (cordycepin, COR) is a challenge because they are very similar in chemical structure. In some previous studies on food ingredients, adenine and dA might be mistakenly detected as COR that has been shown to have multiple health benefits. In this work, we developed a novel HPLC method with fluorescence detction (HPLC-FD) to simultaneously quantify COR, adenosine and dA. Pre-column derivatization with chloroacetaldehyde (CAA) was deployed. The proposed method has a limit of detection at the nM level for COR and adenosine, and is far more sensitive than the methods previously deveopled for COR determination. Using the present method, caterpillar fungi were analyzed as model food samples. The analysis revealed that COR was present in cordyceps militaris and cordyceps flowers in a concentration range from 0.314 to 0.735 mg/g, but not in cordyceps sinensis (C. sinensis), a natural and the priciest caterpillar fungus. These results suggest that the profile of active ingredients in C. sinensis has been wrongly claimed for many years. This finding was also supported by the results from further HPLC-MS/MS analyses.

Direct Analysis of Biofluids by Mass Spectrometry with Microfluidic Voltage-Assisted Liquid Desorption Electrospray Ionization.

Signal suppression by sample matrix in direct electrospray ionization-mass spectrometric (ESI-MS) analysis hampers its clinical and biomedical applications. We report herein the development of a microfluidic voltage-assisted liquid desorption electrospray ionization (VAL-DESI) source to overcome this limitation. Liquid DESI is achieved for the first time in a microfluidic format. Direct analysis of urine, serum, and cell lysate samples by using the proposed microfluidic VAL-DESI-MS/MS method to detect chemical compounds of biomedical interest, including nucleosides, monoamines, amino acids, and peptides is demonstrated. Analyzing a set of urine samples spiked with dihydroxyphenylalanine (DOPA) showed that the assay had a linear calibration curve with r2 value of 0.997 and a limit of detection of 0.055 µM DOPA. The method was applied to simultaneous quantification of nucleosides, that is, cytidine, adenosine, uridine, thymidine, and guanosine in cell lysates using 8-bromoadenosine as internal standard. Adenosine was found most abundant at 26.5 ± 0.57 nmol/106 cells, while thymidine was least at 3.1 ± 0.31 nmol/106 cells. Interestingly, the ratio of adenosine to deoxyadenosine varied significantly from human red blood cells (1.07 ± 0.06) to cancerous cells, including lymphoblast TK6 (0.52 ± 0.02), skin melanoma C32 (0.82 ± 0.04), and promyelocytic leukemia NB4 cells (0.38 ± 0.06). These results suggest that the VAL-DESI-MS/MS technique has a good potential in direct analysis of biofluids. Further, because of the simplicity in its design and operation, the proposed microfluidic liquid DESI source can be fabricated as a disposable device for point-of-care measurements.

Profiling metals in Cordyceps sinensis by using inductively coupled plasma mass spectrometry.

Cordyceps sinensis (C. sinensis) is a natural product that has diverse nutritional and medicinal values. Since the availability of natural C. sinensis becomes limited its authentication and quality control is of high significance. Herein we report on profiling of metals in C. sinensis by using inductively coupled plasma mass spectrometry (ICP-MS). The analysis reveals that C. sinensis contains a wide array of essential elements, including P, Mg, Zn, Cu, Fe, etc. Toxic metals detected are Cd, Pb, and As. In all five samples analyzed Pb contents are below 2.0 ppm. Arsenic level in C. sinensis caterpillar is significantly higher than that in its mycelium and varies from 3.0 to 32 ppm likely due to soil contamination. It's for the first time demonstrated in this work that clustering analysis on the proposed metal profiles consisting of 24 elements is very useful to identify "abnormal" C. sinensis samples, thus adding another dimension to the effective means for authentication and quality assessment of this highly demanded previous natural product.

A microchip electrophoresis-mass spectrometric platform with double cell lysis nano-electrodes for automated single cell analysis.

Capillary electrophoresis-based single cell analysis has become an essential approach in researches at the cellular level. However, automation of single cell analysis has been a challenge due to the difficulty to control the number of cells injected and the irreproducibility associated with cell aggregation. Herein we report the development of a new microfluidic platform deploying the double nano-electrode cell lysis technique for automated analysis of single cells with mass spectrometric detection. The proposed microfluidic chip features integration of a cell-sized high voltage zone for quick single cell lysis, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Built upon this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) has been developed for automated single cell analysis. In the method, cell introduction, cell lysis, and MCE-MS separation are computer controlled and integrated as a cycle into consecutive assays. Analysis of large numbers of individual PC-12 neuronal cells (both intact and exposed to 25mM KCl) was carried out to determine intracellular levels of dopamine (DA) and glutamic acid (Glu). It was found that DA content in PC-12 cells was higher than Glu content, and both varied from cell to cell. The ratio of intracellular DA to Glu was 4.20±0.8 (n=150). Interestingly, the ratio drastically decreased to 0.38±0.20 (n=150) after the cells are exposed to 25mM KCl for 8min, suggesting the cells released DA promptly and heavily while they released Glu at a much slower pace in response to KCl-induced depolarization. These results indicate that the proposed MCE-MS analytical platform may have a great potential in researches at the cellular level.