Comparative analysis of medicinal plants Scutellaria baicalensis and common adulterants based on chloroplast genome sequencing.

BACKGROUND: Scutellaria baicalensis Georgi has been extensively used as a medicinal herb in China for over 2000 years. They may be intentionally or inadvertently substituted or blended with comparable species in the local market, threatening clinical medication safety. Molecular markers are effective tools to prevent misidentification and eliminate doping and falsification among Scutellaria plants. This study screened four highly variable regions to identify Scutellaria and its adulterants. In addition, a phylogenetic analysis was performed using the complete cp genome combined with published Scutellaria species samples. Moreover, a comparative analysis of the cp genomes was conducted to investigate the cp genome evolution of S. baicalensis. RESULTS: The complete cp genome of five species of Scutellaria was sequenced for the first time, and four previously published Scutellaria species were re-sequenced. They all exhibited a conserved quadripartite structure in their cp genomes, including two distinct regions, namely a small and large single copy region, respectively, and two inverted repeats encompassing the majority of ribosomal RNA genes. Furthermore, the nine species exhibited high conservation from aspects of the genome structure, codon usage, repeat sequences, and gene content. Four highly variable regions (matK-rps16, ndhC-trnV-UAC, psbE-petL, and rps16-trnQ-UUG) may function as potential molecular markers for differentiating S. baicalensis from its adulterants. Additionally, the monophyly of Scutellaria was ascertained and could be reclassified into two subgenera, subgenus Anaspis and subgenus Scutellaria, as evidenced by the phylogenetic analyses on sequences of cp genome and shared protein-coding sequences. According to the molecular clock analysis, it has been inferred that the divergence of Scutellaria occurred at approximately 4.0 Mya during the Pliocene Epoch. CONCLUSION: Our study provides an invaluable theoretical basis for further Scutellaria species identification, phylogenetics, and evolution analysis.

Pharmacokinetics and molecular docking of the cardioprotective flavonoids in Dalbergia odorifera.

The purpose of this research was to investigate the cardioprotective effects and pharmacokinetics of Dalbergia odorifera flavonoids. The cardioprotective effects were detected by hematoxylin-eosin staining histopathological observations and the detection of myocardial enzymes by kits in serum, peroxidation and antioxidant levels and ATPase activities by kits in the homogenate supernatant, and antioxidant and apoptosis-related protein expression in heart tissue by immunohistochemistry. The pharmacokinetics parameters of the flavonoids in rat plasma were investigated by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. Molecular docking of the compounds absorbed by the blood with specific proteins was carried out. D. odorifera flavonoids significantly reduced the levels of creatinine kinase, alanine transaminase, nitric oxide, and Hydrogen peroxide, elevated the levels of glutathione, superoxide dismutase, and ATPase, significantly reduced the pathological degree of heart tissue and had obvious anti-myocardial ischemia efficacy. Nine out of the 17 flavonoids were detected in rat plasma. The peak concentration and the area under the plasma concentration-time curve values of 3'-O-methylviolanone and sativanone were significantly higher than those of other ingredients. The peak time of most flavonoids (except for Genistein and Pruneion) was lower than 2 h, while the half-life of elimination of the nine flavonoids ranged from 3.32 to 21.5 h. The molecular docking results showed that daidzein, dalbergin, formononetin, and genistein had the potential to bind to the target proteins. The results of the study provide an important basis for understanding the cardioprotective effects and clinical application of D. odorifera.

Establishment of a rapid and sensitive ic-ELISA for the detection of thiacloprid residues in honey and medicinal herbs using a novel highly specific monoclonal antibody.

Thiacloprid is one of the first generation of neonicotinoid insecticide with a chloropyridine structure like imidacloprid and acetamiprid. Recent studies have revealed its environmental and non-target organism toxicity, leading to restrictions on its use in many countries and regions. Despite limitations, thiacloprid has been detected in various environmental samples, food sources, and biological specimens, posing a significant threat to human health, necessitating advanced detection methods for monitoring. In this study, a highly specific monoclonal antibody against thiacloprid via a multi-immunogen strategy was prepared and a rapid and sensitive enzyme-linked immunosorbent assay for the detection of thiacloprid residues in honey and medicinal herbs was established. The half maximal inhibitory concentration (IC50) of this method was 0.38 ng/mL, improving the sensitivity by 1.2-480.6 times compared to existing reports, and the limit of detection (IC20) was 0.097 ng/mL. The method was successfully applied to the determination of thiacloprid residues in honey and medicinal herbs (Crataegi fructus, Citri reticulatae pericarpium), achieving recovery rates ranging from 87.50 % to 116.11 %. The obtained results were verified using the LC-MS/MS method. The multi-immunogen strategy proposed in this study provides an approach for the preparation of highly sensitive and specific monoclonal antibodies, and immunoassay established based on it has good application prospects in complex matrices.

Application of DNA barcodes in the genetic diversity of hard ticks (Acari: Ixodidae) in Kazakhstan.

Forty-five tick species have been recorded in Kazakhstan. However, their genetic diversity and evolutionary relationships, particularly when compared to ticks in neighbouring countries, remain unclear. In the present study, 148 mitochondrial cytochrome c oxidase subunit I (COI) sequence data from our laboratory and NCBI (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/ ) data were used to address this knowledge gap. Phylogenetic analyses showed that i) Hyalomma anatolicum anatolicum (Koch, 1844) ticks from Jambyl Oblast (southeastern Kazakhstan) and Gansu Province (northwestern China) constituted a newly deviated clade; and ii) Dermacentor reticulatus (Fabricius, 1974) ticks from South Kazakhstan Oblast were closer to those in Romania and Turkey. The network diagram of haplotypes showed that i) the H-1 and H-2 haplotypes of Dermacentor marginatus (Sulzer, 1776) ticks from Zhetisu and Almaty were all newly evolved; and ii) the H-3 haplotypes of Haemaphysalis erinacei (Pavesi, 1884) from Almaty Oblast and Xinjiang Uygur Autonomous Region (northwestern China) were evolved from the H-1 haplotype from Italy. In the future, more COI data from different tick species, especially from Kazakhstan and neighbouring countries, should be employed in the field of tick DNA barcoding.

Analysis of codon usage patterns in 48 Aconitum species.

BACKGROUND: The Aconitum genus is a crucial member of the Ranunculaceae family. There are 350 Aconitum species worldwide, with about 170 species found in China. These species are known for their various pharmacological effects and are commonly used to treat joint pain, cold abdominal pain, and other ailments. Codon usage bias (CUB) analysis contributes to evolutionary relationships and phylogeny. Based on protein-coding sequences (PCGs), we selected 48 species of Aconitum for CUB analysis. RESULTS: The results revealed that Aconitum species had less than 50% GC content. Furthermore, the distribution of GC content was irregular and followed a trend of GC1 > GC2 > GC3, indicating a bias towards A/T bases. The relative synonymous codon usage (RSCU) heat map revealed the presence of conservative codons with slight variations within the genus. The effective number of codons (ENC)-Plot and the parity rule 2 (PR2)-bias plot analysis indicate that natural selection is the primary factor influencing the variation in codon usage. As a result, we screened various optimal codons and found that A/T bases were preferred as the last codon. Furthermore, our Maximum Likelihood (ML) analysis based on PCGs among 48 Aconitum species yielded results consistent with those obtained from complete chloroplast (cp.) genome data. This suggests that analyzing mutation in PCGs is an efficient method for demonstrating the phylogeny of species at the genus level. CONCLUSIONS: The CUB analysis of 48 species of Aconitum was mainly influenced by natural selection. This study reveals the CUB pattern of Aconitum and lays the foundation for future genetic modification and phylogenetic analyses.

Comparative transcriptome analysis reveals the regulatory mechanisms of two tropical water lilies in response to cold stress.

BACKGROUND: Tropical water lily is an aquatic plant with high ornamental value, but it cannot overwinter naturally at high latitudes. The temperature drop has become a key factor restricting the development and promotion of the industry. RESULTS: The responses of Nymphaea lotus and Nymphaea rubra to cold stress were analyzed from the perspective of physiology and transcriptomics. Under the cold stress, Nymphaea rubra had obvious leaf edge curling and chlorosis. The degree of peroxidation of its membrane was higher than that of Nymphaea lotus, and the content of photosynthetic pigments also decreased more than that of Nymphaea lotus. The soluble sugar content, SOD enzyme activity and CAT enzyme activity of Nymphaea lotus were higher than those of Nymphaea rubra. This indicated that there were significant differences in the cold sensitivity of the two varieties. GO enrichment and KEGG pathway analysis showed that many stress response genes and pathways were affected and enriched to varying degrees under the cold stress, especially plant hormone signal transduction, metabolic pathways and some transcription factor genes were from ZAT gene family or WKRY gene family. The key transcription factor ZAT12 protein in the cold stress response process has a C2H2 conserved domain, and the protein is localized in the nucleus. Under the cold stress, overexpression of the NlZAT12 gene in Arabidopsis thaliana increased the expression of some cold-responsive protein genes. The content of reactive oxygen species and MDA in transgenic Arabidopsis thaliana was lower, and the content of soluble sugar was higher, indicating that overexpression of NlZAT12 can improve the cold tolerance of Arabidopsis thaliana. CONCLUSION: We demonstrate that ethylene signalling and reactive oxygen species signalling play critical roles in the response of the two cultivars to cold stress. The key gene NlZAT12 for improving cold tolerance was identified. Our study provides a theoretical basis for revealing the molecular mechanism of tropical water lily in response to cold stress.

DNA Metabarcoding Reveals the Fungal Community on the Surface of Lonicerae Japonicae Flos, an Edible and Medicinal Herb.

Lonicerae Japonicae Flos (LJF) has been globally applied as an herbal medicine and tea. A number of reports recently revealed fungal and mycotoxin contamination in medicinal herbs. It is essential to analyze the fungal community in LJF to provide an early warning for supervision. In this study, the fungal community in LJF samples was identified through DNA metabarcoding. A total of 18 LJF samples were collected and divided based on the collection areas and processing methods. The results indicated that Ascomycota was the dominant phylum. At the genus level, Rhizopus was the most abundant, followed by Erysiphe and Fusarium. Ten pathogenic fungi were detected among the 41 identified species. Moreover, Rhizopus, Fusarium, and Aspergillus had lower relative abundances in LJF samples under oven drying than under other processing methods. This work is expected to provide comprehensive knowledge of the fungal community in LJF and a theoretical reference for enhanced processing methods in practical manufacturing.

Anti-HIV Potential of Beesioside I Derivatives as Maturation Inhibitors: Synthesis, 3D-QSAR, Molecular Docking and Molecular Dynamics Simulations.

HIV-1 maturation is the final step in the retroviral lifecycle that is regulated by the proteolytic cleavage of the Gag precursor protein. As a first-in-class HIV-1 maturation inhibitor (MI), bevirimat blocks virion maturation by disrupting capsid-spacer peptide 1 (CA-SP1) cleavage, which acts as the target of MIs. Previous alterations of beesioside I (1) produced (20S,24S)-15ꞵ,16ꞵ-diacetoxy-18,24; 20,24-diepoxy-9,19-cyclolanostane-3ꞵ,25-diol 3-O-3',3'-dimethylsuccinate (3, DSC), showing similar anti-HIV potency compared to bevirimat. To ascertain the binding modes of this derivative, further modification of compound 1 was conducted. Three-dimensional quantitative structure−activity relationship (3D-QSAR) analysis combined with docking simulations and molecular dynamics (MD) were conducted. Five new derivatives were synthesized, among which compound 3b showed significant activity against HIV-1NL4-3 with an EC50 value of 0.28 µM. The developed 3D-QSAR model resulted in great predictive ability with training set (r2 = 0.99, q2 = 0.55). Molecular docking studies were complementary to the 3D-QSAR analysis, showing that DSC was differently bound to CA-SP1 with higher affinity than that of bevirimat. MD studies revealed that the complex of the ligand and the protein was stable, with root mean square deviation (RMSD) values <2.5 Å. The above results provided valuable insights into the potential of DSC as a prototype to develop new antiviral agents.

Insights into the intestinal toxicity of foodborne mycotoxins through gut microbiota: A comprehensive review.

Mycotoxins, which are fungal metabolites, pose a significant global food safety concern by extensively contaminating food and feed, thereby seriously threatening public health and economic development. Many foodborne mycotoxins exhibit potent intestinal toxicity. However, the mechanisms underlying mycotoxin-induced intestinal toxicity are diverse and complex, and effective prevention or treatment methods for this condition have not yet been established in clinical and animal husbandry practices. In recent years, there has been increasing attention to the role of gut microbiota in the occurrence and development of intestinal diseases. Hence, this review aims to provide a comprehensive summary of the intestinal toxicity mechanisms of six common foodborne mycotoxins. It also explores novel toxicity mechanisms through the "key gut microbiota-key metabolites-key targets" axis, utilizing multiomics and precision toxicology studies with a specific focus on gut microbiota. Additionally, we examine the potential beneficial effects of probiotic supplementation on mycotoxin-induced toxicity based on initial gut microbiota-mediated mycotoxicity. This review offers a systematic description of how mycotoxins impact gut microbiota, metabolites, and genes or proteins, providing valuable insights for subsequent toxicity studies of mycotoxins. Furthermore, it lays a theoretical foundation for preventing and treating intestinal toxicity caused by mycotoxins and advancing food safety practices.

[Mercury species analysis and tissue distribution in rats after continuous administration of Cinnabaris].

Cinnabaris is a traditional Chinese medicine(TCM) commonly used for sedation and tranquilization in clinics, and its safety has always been a concern. This study intends to investigate the species and tissue distribution of mercury in rats after continuous administration of Cinnabaris. In the experiment, 30 rats were randomly divided into the control group(equivalent to 0.5% carboxy-methyl cellulose sodium), low-dose Cinnabaris group(0.2 g·kg~(-1)), high-dose Cinnabaris group(2 g·kg~(-1)), pseudogerm-free control group(equivalent to 0.5% sodium carboxymethyl cellulose), and pseudogerm-free Cinnabaris group(2 g·kg~(-1)). They were orally administered for 30 consecutive days. Ultrasound-assisted acid extraction method combined with high performance liquid chromatography and inductively coupled plasma-mass spectrometry(HPLC-ICP-MS) was adopted to determine inorganic mercury [Hg(Ⅱ)], methylmercury(MeHg), and ethylmercury(EtHg) in different tissue, plasma, urine, and feces of rats. The optimal detection conditions and extraction methods were optimized, and the linearity(R~2>0.999 3), precision(RSD<7.0%), and accuracy(spike recoveries ranged from 73.05% to 109.5%) of all the mercury species were satisfied, meeting the requirements of analysis. The results of mercury species detection showed that Hg(Ⅱ) was detected in all the tissue of the five experimental groups, and the main accumulating organs were the intestinal tract, stomach, and kidney. MeHg existed at a low concentration in most tissue, and EtHg was not detected in all groups. In addition, pathological examination results showed that hepatocyte vacuolar degeneration, loose cytoplasm, light staining, and mononuclear cell infiltration were observed in the high-dose Cinnabaris group, low-dose Cinnabaris group, and pseudogerm-free Cinnabaris group, with slightly milder lesions in the low-dose Cinnabaris group. Hydrous degeneration of renal tubular epithelium could be seen in the high-dose Cinnabaris group and pseudogerm-free Cinnabaris group, but there was no significant difference between the other groups and the control group. No abnormal changes were found in the brain tissue of rats in each group. This paper studied the different mercury species and tissue distribution in normal and pseudogerm-free rats after continuous administration of Cinnabaris for 30 days and clarified its effects on the tissue structure of the liver, kidney, and brain, which provided supporting evidence for the safety evaluation of Cinnabaris.

Intravesical Pseudomonas aeruginosa mannose-sensitive Hemagglutinin vaccine triggers a tumor-preventing immune environment in an orthotopic mouse bladder cancer model.

Bacillus Calmette-Guerin (BCG) immunotherapy can prevent recurrence and progression in selected patients with non-muscle-invasive bladder cancer (NMIBC); however, significant adverse events and treatment failure suggest the need for alternative agents. A commercial anti-infection vaccine comprises a genetically engineered heat-killed Pseudomonas aeruginosa (PA) expressing many mannose-sensitive hemagglutination (MSHA) fimbriae, termed PA-MSHA, which could be a candidate for bladder cancer intravesical therapy. In an immunocompetent orthotopic MB49 bladder cancer model, we characterized the antitumor effects and mechanisms of PA-MSHA compared with those of BCG. Three weekly intravesical PA-MSHA or BCG treatments reduced tumor involvement; however, only PA-MSHA prolonged survival against MB49 implantation significantly. In non-tumor-bearing mice after treatment, flow-cytometry analysis showed PA-MSHA and BCG induced an increased CD4/CD8 ratio, the levels of effector memory T cell phenotypes (CD44, CXCR-3, and IFN-γ), and the proportion of CD11b+Ly6G-Ly6C-IA/IE+ mature macrophages, but a decrease in the proportion of CD11b+Ly6G-Ly6C+IA/IE- monocytic myeloid-derived suppressor cells (Mo-MDSCs) and the expression of suppressive molecules on immune cells (PD-L1, PD-1, TIM-3, and LAG-3). Notably, PA-MSHA, but not BCG, significantly reduced PD-1 and TIM-3 expression on CD4+ T cells, which might account for the better effects of PA-MSHA than BCG. However, in tumor-bearing mice after treatment, the increased proportion of Mo-MDSCs and high expression of PD-L1 might be involved in treatment failure. Thus, modulating the balance among adaptive and innate immune responses was identified as a key process underlying PA-MSHA-mediated treatment efficacy. The results demonstrated mechanisms underlying intravesical PA-MSHA therapy, pointing at its potential as an alternative effective treatment for NMIBC.

Hepatotoxicity of food-borne mycotoxins: molecular mechanism, anti-hepatotoxic medicines and target prediction.

Mycotoxins are metabolites produced by fungi. The widespread contamination of food and feed by mycotoxins is a global food safety problem and a serious threat to people's health. Most food-borne mycotoxins have strong hepatotoxicity. However, no effective methods have been found to prevent or treat Mycotoxin- Induced Liver Injury (MILI) in clinical and animal husbandry. In this paper, the molecular mechanisms and potential anti-MILI medicines of six food-borne MILI are reviewed, and their targets are predicted by network toxicology, which provides a theoretical basis for further study of the toxicity mechanism of MILI and the development of effective strategies to manage MILI-related health problems in the future and accelerate the development of food safety.

The presence and transfer characteristics of aflatoxins in medicinal herbs: From raw materials to edible dispensing granules.

In this study, a sensitive and accurate immunoaffinity columns coupled with high-performance liquid chromatography method was established to monitor the presence of aflatoxins-aflatoxin B1 , aflatoxin B2 , aflatoxin G1 , and aflatoxin G2 -in different medicinal herbs. The proposed method was found to be suitable for the detection of aflatoxins in eight kinds of herbs and their corresponding granules. Two batches of Arecae semen were positive for aflatoxins, with high residue levels of different aflatoxins. To better understand the presence and transfer of aflatoxins during the formulation of dispensing granules from the herbs, the aflatoxins-free herbs were artificially inoculated with Aspergillus flavus to explore aflatoxins production. Both aflatoxin B1 and aflatoxin B2 were detected in all inoculated samples, while aflatoxin G2 was only detected in Astragali radix samples. Additionally, the presence of aflatoxin B1 was extremely high compared to other aflatoxins. More specifically, the transfer rate of the aflatoxin B1 and the total aflatoxins from original herbs to granules were both approximately 40%. These findings indicated that the preparation of herbs into dispensing granules reduced the content of aflatoxins. The high-level presence of aflatoxins in inoculated herbs indicated that attention is needed to the safety of A. flavus-contaminated herbs.

Further insight into the potential toxicity of zearalenone-14-glucoside based on toxicokinetics, tissue distribution, transformation, and excretion in rats.

Bioaccumulation and biotransformation are critical factors that affect the release of easily metabolizable chemicals to cause human toxicity. The glucoside-type modified mycotoxin Zearalenone-14-Glucoside (Z14G) has attracted global attention for its high occurrence in foodstuffs and the potential threat to humans as its high rate of transformation into parent forms. Given the limited toxicokinetics information, this study assessed the absorption, distribution, biotransformation and excretion of Z14G, aiming to define the potential risk of Z14G. The toxicokinetics of Z14G were assessed after intravenous (IV) or oral administration (PO) in SD rats at doses of 10 mg/kg·b.w. In addition, comparative work with the parent mycotoxin ZEN was performed in parallel. The determination of Z14G and its metabolites (ZEN, α-zearalenol, ß-zearalenol, α-zearalanol, ß-zearalanol) proceeded with a sensitive UHPLC-MS/MS method. Our research indicated that Z14G readily disappeared from the blood, and distributed throughout the tissues via transformation into its parent form ZEN, and excreted primarily through urine. More importantly, the metabolite α-ZEL was observed in most analyzed tissue, urine and feces samples. Overall, our findings highlight the importance of biotransformation with regard to Z14G, providing critical insight for the health risk assessment of co-exposure of humans to glucoside-type modified mycotoxins.

A highly parallel DTT/MB-DNA/Au electrochemical biosensor for trace Hg monitoring by using configuration occupation approach and SECM.

Environmental pollution and medicine safety have aroused increasing public concerns due to human health. Amongst various contaminants, mercury is of special attention owing to their environmental persistence and biogeochemical recycling and ecological risks. Herein, a simple and highly parallel electrochemical biosensor for Hg determination was designed and investigated. The proposed biosensor was prepared and compared between (1) DTT/MB-DNA/Au with configuration occupation approach and (2) MCH/MB-DNA/Au with passivation approach. According to the combined results of scanning electrochemical microscope (SECM) and Randles-Sevcik equation, the DTT modified electrode exhibited high uniformity on DNA distribution and superb stability on electron transfer in Hg2+ detection. Evidentially, the response value of proposed DTT/MB-DNA/Au was increased from 57.518% to 97.607%, while RSD% between duplicate runs had dropped from 22.658% to 0.223% (n = 3). Moreover, the increased proportion of effective working area was 467.380% compared with general sensors. Besides, DTT concentration, DNA concentration as well as assembly time were optimized, utilizing electrochemical impedance spectroscopy (EIS), Cyclic Voltammetry (CV) and Square Wave Anode Stripping Voltammetry (SWASV). This optimized biosensor exhibited an excellent selectivity toward Hg2+ over Cu2+, As2+, Cd2+, Pb2+, Cr3+, Ni2+ and Zn2+ etc., and the stability of DTT/MB-DNA/Au were at least two times better even after 3 days under room temperature. Also, a linear relation was observed between the peak current and Hg2+concentrations in a range from 0.25 nM to 2.00 µM with a detection limit of 53.00 pM under optimal conditions. Finally, DTT/MB-DNA/Au was applied for plants and medical products analysis. In all, this optimized DTT/MB-DNA/Au with advantages of high repeatability and sensitivity would provide a new insight into the design and application of biosensor for reliable sensing in safeguarding plant protection and medicinal safety.

[Residual status, toxicity, and analytical method of banned pesticides in traditional Chinese medicines].

A total of 33 pesticides have been banned from Chinese medicinal materials and decoction pieces(plants) according to Chinese Pharmacopoeia(2020 edition). According to the chemical structures, they are mainly divided into seven categories: organophosphorus compounds, organochlorines, carbamates, amidines, sulfonylureas, phenylpyrazoles, and ethers. These banned pesticides exhibit neurotoxicity, reproductive toxicity, immune system toxicity, teratogenicity, carcinogenesis, and mutagenesis, seriously damaging human and animal health. They affect not only the quality and safety of traditional Chinese medicines and resulting products, but also their competitiveness in the international market. Due to the numerous varieties of traditional Chinese medicines and their complex substrates, it is necessary to establish a universal and highly sensitive method for pesticide residue detection. This review systematically summarized the residual status, toxicity, and analytical methods of banned pesticides in traditional Chinese medicines, and forecasted the prospects of different analytical techniques, so as to provide reference for further safety and risk assessment of banned pesticide residues in traditional Chinese medicines, thus ensuring the safe production of traditional Chinese medicines.

Human Tacheng Tick Virus 2 Infection, China, 2019.

We used metagenomic analysis to identify Tacheng tick virus 2 infection in a patient with a history of tick bite in northwestern China. We confirmed the virus with reverse transcription-PCR, virus isolation, and genomic analysis. We detected viral RNA in 9.6% of ticks collected from the same region.

Development and validation of a PD-L1/PD-1/CD8 axis-based classifier to predict cancer survival of upper tract urothelial carcinoma after radical nephroureterectomy.

The expression status of programmed cell death-ligand 1/programmed cell death 1 (PD-L1/PD-1) and the infiltration of CD8+ T cells in tumor tissues are considered to be related to immunotherapy efficacy and patient prognosis. The purpose of this study is to clarify the prognostic value of the PD-L1/PD-1/CD8 axis, and to develop and validate a comprehensive scoring system based on multiple immune variables to predict cancer survival of upper tract urothelial carcinoma (UTUC) after radical nephroureterectomy (RNU). The immunohistochemical method was used to detect the expression of PD-L1, PD-1, and CD8 in cancer tissues of UTUC patients after RNU. Then, an immunoscore was constructed using the least absolute shrinkage and selection operator (LASSO) Cox regression model in the training cohort (n = 120), and it was verified in the validation cohort (n = 54). We found that infiltration of PD-L1+ immune cells (ICs), stromal PD-1+ tumor-infiltrating lymphocytes (TILs), and intratumoral CD8+ TILs was associated with poor overall survival (OS). The immunoscore based on the three immune variables further divided the patients into low- and high-risk groups, and there was a significant difference in the survival rate. A nomogram was constructed by combining tumor-node-metastasis (TNM) stage and immunoscore, and the area under the curve of the receiver-operating characteristic (ROC) (0.78) for predicting 5-year mortality was better than that of the TNM stage (0.70) and immunoscore (0.76). Our results show that the PD-L1/PD-1/CD8 axis-based classifier have potential clinical application to predict cancer survival of UTUC patients after RNU.

Mitochondrial GRIM-19 deficiency facilitates gastric cancer metastasis through oncogenic ROS-NRF2-HO-1 axis via a NRF2-HO-1 loop.

BACKGROUND: NRF2, a prime target of cellular defense against oxidative stress, has shown a dark side profile in cancer progression. GRIM-19, an essential subunit of the mitochondrial MRC complex I, was recently identified as a suppressive role in tumorigenesis of human gastric cancer (GC). However, little information is available on the role of GRIM-19 and its cross-talk with NRF2 in GC metastasis. METHODS: Online GC database was used to investigate DNA methylation and survival outcomes of GRIM-19. CRISPR/Cas9 lentivirus-mediated gene editing, metastasis mice models and pharmacological intervention were applied to investigate the role of GRIM-19 deficiency in GC metastasis. Quantitative RT-PCR, FACS, Western blot, IHC, IF and reporter gene assay were performed to explore underlying mechanisms. RESULTS: Low GRIM-19 is correlated with poor survival outcome of GC patients while DNA hypermethylation is associated with GRIM-19 downregulation. GRIM-19 deficiency facilitates GC metastasis and triggers aberrant oxidative stress as well as ROS-dependent NRF2-HO-1 activation. Experimental interventions of specific ROS, NRF2 or HO-1 inhibitor significantly abrogate GRIM-19 deficiency-driven GC metastasis in vitro and in vivo. Moreover, HO-1 inhibition not only reverses GRIM-19 deficiency-driven NRF2 activation, but also feedback blocks NRF2 activator-induced NRF2 signaling, resulting in decreased metastasis-associated genes. CONCLUSIONS: Our data suggest that GRIM-19 deficiency accelerates GC metastasis through the oncogenic ROS-NRF2-HO-1 axis via a positive-feedback NRF2-HO-1 loop. Therefore, this study not only offers novel insights into the role of oncogenic NRF2 in tumor progression, but also provides new strategies to alleviate the dark side of NRF2 by targeting HO-1.

Colloidal gold-based lateral flow immunoassay with inline cleanup for rapid on-site screening of carbendazim in functional foods.

In this study, for the first time, we propose a sensitive colloidal gold-based lateral flow immunoassay (LFIA) that can be used to detect carbendazim residues in functional foods. The adoption of inline cleanup LFIA strips effectively improved background interference to reduce misjudgment of results. First, the hapten 2-(methylamino)-1H-benzo[d]imidazole-5-carboxylic acid was used to establish the carbendazim immunoassay method. Subsequently, colloidal gold-mAb preparation and LFIA detection conditions were systematically optimized. For root and fruit samples (ginseng, ginger, jujube, and Chinese wolfberry), the designed strips had a cutoff value of 8 ng/mL. For flower and seed samples (chrysanthemum, coix seed, and malt), the cutoff value was 12 ng/mL. Even in a complex matrix, the established LFIA method demonstrates satisfactory sensitivity and anti-interference ability. This method was successfully applied in detection of carbendazim residues in complex functional foods, and the assay results are consistent with those obtained via liquid chromatography-tandem mass spectrometry. In short, the proposed method is fast and sensitive and has strong anti-interference ability. Furthermore, it provides a new technical method highly relevant to the on-site rapid detection of carbendazim residues in complex sample matrix.