Journal of Pharmaceutical Analysis

Evaluation of immunoprotection against coronavirus disease 2019: Novel variants, vaccine inoculation, and complications

Congshan Jiang, Kaichong Jiang, Xiaowei Li, Ning Zhang, Wenhua Zhu, Liesu Meng, Yanmin Zhang, Shemin Lu

2023, 13(1): 1-10. doi: 10.1016/j.jpha.2022.10.003

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Abstract:
The strikingly rapidly mutating nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome has been a constant challenge during the coronavirus disease 2019 (COVID-19) pandemic. In this study, various techniques, including reverse transcription-quantitative polymerase chain reaction, antigen-detection rapid diagnostic tests, and high-throughput sequencing were analyzed under different scenarios and spectra for the etiological diagnosis of COVID-19 at the population scale. This study aimed to summarize the latest research progress and provide up-to-date understanding of the methodology used for the evaluation of the immunoprotection conditions against future variants of SARS-CoV-2. Our novel work reviewed the current methods for the evaluation of the immunoprotection status of a specific population (endogenous antibodies) before and after vaccine inoculation (administered with biopharmaceutical antibody products). The present knowledge of the immunoprotection status regarding the COVID-19 complications was also discussed. Knowledge on the immunoprotection status of specific populations can help guide the design of pharmaceutical antibody products, inform practice guidelines, and develop national regulations with respect to the timing of and need for extra rounds of vaccine boosters.

Inosine: A broad-spectrum anti-inflammatory against SARS-CoV-2 infection-induced acute lung injury via suppressing TBK1 phosphorylation

Ningning Wang, Entao Li, Huifang Deng, Lanxin Yue, Lei Zhou, Rina Su, Baokun He, Chengcai Lai, Gaofu Li, Yuwei Gao, Wei Zhou, Yue Gao

2023, 13(1): 11-23. doi: 10.1016/j.jpha.2022.10.002

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Abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storms constitute the primary cause of coronavirus disease 19 (COVID-19) progression, severity, criticality, and death. Glucocorticoid and anti-cytokine therapies are frequently administered to treat COVID-19, but have limited clinical efficacy in severe and critical cases. Nevertheless, the weaknesses of these treatment modalities have prompted the development of anti-inflammatory therapy against this infection. We found that the broad-spectrum anti-inflammatory agent inosine downregulated proinflammatory interleukin (IL)-6, upregulated anti-inflammatory IL-10, and ameliorated acute inflammatory lung injury caused by multiple infectious agents. Inosine significantly improved survival in mice infected with SARS-CoV-2. It indirectly impeded TANK-binding kinase 1 (TBK1) phosphorylation by binding stimulator of interferon genes (STING) and glycogen synthase kinase-3β (GSK3β), inhibited the activation and nuclear translocation of the downstream transcription factors interferon regulatory factor (IRF3) and nuclear factor kappa B (NF-κB), and downregulated IL-6 in the sera and lung tissues of mice infected with lipopolysaccharide (LPS), H1N1, or SARS-CoV-2. Thus, inosine administration is feasible for clinical anti-inflammatory therapy against severe and critical COVID-19. Moreover, targeting TBK1 is a promising strategy for inhibiting cytokine storms and mitigating acute inflammatory lung injury induced by SARS-CoV-2 and other infectious agents.

Epigenetic drug library screening reveals targeting DOT1L abrogates NAD⁺ synthesis by reprogramming H3K79 methylation in uveal melanoma

Xiang Gu, Yu Hua, Jie Yu, Ludi Yang, Shengfang Ge, Renbing Jia, Peiwei Chai, Ai Zhuang, Xianqun Fan

2023, 13(1): 24-38. doi: 10.1016/j.jpha.2022.11.008

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Abstract:
Uveal melanoma (UM) is the most frequent and life-threatening ocular malignancy in adults. Aberrant histone methylation contributes to the abnormal transcriptome during oncogenesis. However, a comprehensive understanding of histone methylation patterns and their therapeutic potential in UM remains enigmatic. Herein, using a systematic epi-drug screening and a high-throughput transcriptome profiling of histone methylation modifiers, we observed that disruptor of telomeric silencing-1-like (DOT1L), a methyltransferase of histone H3 lysine 79 (H3K79), was activated in UM, especially in the high-risk group. Concordantly, a systematic epi-drug library screening revealed that DOT1L inhibitors exhibited salient tumor-selective inhibitory effects on UM cells, both in vitro and in vivo. Combining Cleavage Under Targets and Tagmentation (CUT&Tag), RNA sequencing (RNA-seq), and bioinformatics analysis, we identified that DOT1L facilitated H3K79 methylation of nicotinate phosphoribosyltransferase (NAPRT) and epigenetically activated its expression. Importantly, NAPRT served as an oncogenic accelerator by enhancing nicotinamide adenine dinucleotide (NAD⁺) synthesis. Therapeutically, DOT1L inhibition epigenetically silenced NAPRT expression through the diminishment of dimethylation of H3K79 (H3K79me2) in the NAPRT promoter, thereby inhibiting the malignant behaviors of UM. Conclusively, our findings delineated an integrated picture of the histone methylation landscape in UM and unveiled a novel DOT1L/NAPRT oncogenic mechanism that bridges transcriptional addiction and metabolic reprogramming.

The interaction between polyphyllin I and SQLE protein induces hepatotoxicity through SREBP-2/HMGCR/SQLE/LSS pathway

Zhiqi Li, Qiqi Fan, Meilin Chen, Ying Dong, Farong Li, Mingshuang Wang, Yulin Gu, Simin Guo, Xianwen Ye, Jiarui Wu, Shengyun Dai, Ruichao Lin, Chongjun Zhao

2023, 13(1): 39-54. doi: 10.1016/j.jpha.2022.11.005

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Abstract:
Polyphyllin I (PPI) and polyphyllin II (PII) are the main active substances in the Paris polyphylla. However, liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated. In this work, we found that PPI and PII exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner. The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepatotoxicity of PPI and PII was associated with the cholesterol biosynthetic pathway disorders, which were alleviated by the cholesterol biosynthesis inhibitor lovastatin. Additionally, 3-hydroxy-3-methy-lglutaryl CoA reductase (HMGCR) and squalene epoxidase (SQLE), the two rate-limiting enzymes in the cholesterol synthesis, selected as the potential targets, were confirmed by the molecular docking, the overexpression, and knockdown of HMGCR or SQLE with siRNA. Finally, the pull-down and surface plasmon resonance technology revealed that PPI could directly bind with SQLE but not with HMGCR. Collectively, these data demonstrated that PPI-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways, thus disturbing the cholesterol biosynthesis pathway. The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.

Solid-phase microextraction of endogenous metabolites from intact tissue validated using a Biocrates standard reference method kit

Runshan Will Jiang, Karol Jaroch, Janusz Pawliszyn

2023, 13(1): 55-62. doi: 10.1016/j.jpha.2022.09.002

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Abstract:
Improved analytical methods for the metabolomic profiling of tissue samples are constantly needed. Currently, conventional sample preparation methods often involve tissue biopsy and/or homogenization, which disrupts the endogenous metabolome. In this study, solid-phase microextraction (SPME) fibers were used to monitor changes in endogenous compounds in homogenized and intact ovine lung tissue. Following SPME, a Biocrates AbsoluteIDQ assay was applied to make a downstream targeted metabolomics analysis and confirm the advantages of in vivo SPME metabolomics. The AbsoluteIDQ kit enabled the targeted analysis of over 100 metabolites via solid-liquid extraction and SPME. Statistical analysis revealed significant differences between conventional liquid extractions from homogenized tissue and SPME results for both homogenized and intact tissue samples. In addition, principal component analysis revealed separated clustering among all the three sample groups, indicating changes in the metabolome due to tissue homogenization and the chosen sample preparation method. Furthermore, clear differences in free metabolites were observed when extractions were performed on the intact and homogenized tissue using identical SPME procedures. Specifically, a direct comparison showed that 47 statistically distinct metabolites were detected between the homogenized and intact lung tissue samples (P < 0.05) using mixed-mode SPME fibers. These changes were probably due to the disruptive homogenization of the tissue. This study's findings highlight both the importance of sample preparation in tissue-based metabolomics studies and SPME's unique ability to perform minimally invasive extractions without tissue biopsy or homogenization while providing broad metabolite coverage.

Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Ying Zhang, Qinghua Cai, Yuxiang Luo, Yu Zhang, Huilin Li

2023, 13(1): 63-72. doi: 10.1016/j.jpha.2022.11.003

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Abstract:
Ribosomes are abundant, large RNA-protein complexes that are the sites of all protein synthesis in cells. Defects in ribosomal proteins (RPs), including proteoforms arising from genetic variations, alternative splicing of RNA transcripts, post-translational modifications and alterations of protein expression level, have been linked to a diverse range of diseases, including cancer and aging. Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets. In the present work, using E. coli 70S RPs as an example, we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry (MS) system, and then applied it to the HeLa 80S ribosome. The results were complemented by a bottom-up approach. In total, 50 out of 55 RPs were identified using the top-down approach. Among these, more than 30 RPs were found to have their N-terminal methionine removed. Additional modifications such as methylation, acetylation, and hydroxylation were also observed, and the modification sites were identified by bottom-up MS. In a HeLa 80S ribosomal sample, we identified 98 ribosomal proteoforms, among which multiple truncated 80S ribosomal proteoforms were observed, the type of information which is often overlooked by bottom-up experiments. Although their relevance to diseases is not yet known, the integration of top-down and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

Untargeted and targeted mass spectrometry reveal the effects of theanine on the central and peripheral metabolomics of chronic unpredictable mild stress-induced depression in juvenile rats

Yanru Zhu, Feng Wang, Jiatong Han, Yunli Zhao, Miao Yu, Mingyan Ma, Zhiguo Yu

2023, 13(1): 73-87. doi: 10.1016/j.jpha.2022.10.001

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Abstract:
L-theanine has been shown to have a therapeutic effect on depression. However, whether L-theanine has an excellent preventive effect on depression in children and adolescents and what its mechanism is have not been well explained. Given the complexity of the pathogenesis of depression, this study investigated the preventive effect and mechanism of L-theanine on depression in juvenile rats by combining serum and hippocampal metabolomic strategies. Behavioral tests, hippocampal tissue sections, and serum and hippocampal biochemical indexes were studied, and the results confirmed the preventive effect of L-theanine. Untargeted reversed-phase liquid chromatography-quadrupole-time-of-flight mass spectrometry and targeted hydrophilic interaction liquid chromatography-triple quadrupole mass spectrometry were developed to analyze the metabolism changes in the serum and hippocampus to screen for potential biomarkers related to L-theanine treatment. The results suggested that 28 abnormal metabolites in the serum and hippocampus that were considered as potential biomarkers returned to near-normal levels after L-theanine administration. These biomarkers were involved in various metabolic pathways, mainly including amino acid metabolism and lipid metabolism. The levels of amino acids and neurotransmitters in the phenylalanine, tryptophan, and glutamic acid pathways were significantly reduced after L-theanine administration compared with chronic unpredictable mild stress-induced rats. In summary, L-theanine had a significant preventive effect on depression and achieved its preventive results on depression by regulating various aspects of the body, such as amino acids, lipids, and inflammation. This research systematically analyzed the mechanism of L-theanine in preventing depression and laid the foundation for applying L-theanine to prevent depression in children and adolescents.

Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry

Yingrui Zhang, Shiyu Chen, Fangfang Fan, Ning Xu, Xian-Li Meng, Yi Zhang, Jin-Ming Lin

2023, 13(1): 88-98. doi: 10.1016/j.jpha.2022.11.007

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Abstract:
Aconitine, a common and main toxic component of Aconitum, is toxic to the central nervous system. However, the mechanism of aconitine neurotoxicity is not yet clear. In this work, we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine. HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system. Meanwhile, to confirm the metabolic mechanism of aconitine toxicity on HT22 cells, the levels of lactate dehydrogenase, intracellular Ca²⁺, reactive oxygen species, glutathione and superoxide dismutase, and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology. Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid, which was followed by the accumulation of lactic acid and reduction of glucose. The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca²⁺ overload and oxidative stress, and eventually result in cell apoptosis. In general, we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

Boosting ferroptosis and microtubule inhibition for antitumor therapy via a carrier-free supermolecule nanoreactor

Min Mu, Xiaoyan Liang, Na Zhao, Di Chuan, Bo Chen, Shasha Zhao, Guoqing Wang, Rangrang Fan, Bingwen Zou, Bo Han, Gang Guo

2023, 13(1): 99-109. doi: 10.1016/j.jpha.2022.09.003

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Abstract:
Traditional microtubule inhibitors fail to significantly enhance the effect of colorectal cancer; hence, new and efficient strategies are necessary. In this study, a supramolecular nanoreactor (DOC@TA-Fe³⁺) based on tannic acid (TA), iron ion (Fe³⁺), and docetaxel (DOC) with microtubule inhibition, reactive oxygen species (ROS) generation, and glutathione peroxidase 4 (GPX4) inhibition, is prepared for ferroptosis/apoptosis treatment. After internalization by CT26 cells, the DOC@TA-Fe³⁺ nanoreactor escapes from the lysosomes to release payloads. The subsequent Fe³⁺/Fe²⁺ conversion mediated by TA reducibility can trigger the Fenton reaction to enhance the ROS concentration. Additionally, Fe³⁺ can consume glutathione to repress the activity of GPX4 to induce ferroptosis. Meanwhile, the released DOC controls microtubule dynamics to activate the apoptosis pathway. The superior in vivo antitumor efficacy of DOC@TA-Fe³⁺ nanoreactor in terms of tumor growth inhibition and improved survival is verified in CT26 tumor-bearing mouse model. Therefore, the nanoreactor can act as an effective apoptosis and ferroptosis inducer for application in colorectal cancer therapy.

2023 Vol. 13, No. 1